Antioxidant protein 2, gene and methods of use therefor

ABSTRACT

The present invention involves the identification of a novel gene and protein, now designated as antioxidant protein 2 (Aop2 and AOP2, respectively). Studies indicate that Ltw4 and Aop2 are a single gene. In addition, Aop2 also appears to be the gene responsible for the Athl trait in mice—a predisposition to atherosclerotic disease. The human homolog for this gene also has been identified. This discovery makes possible a variety of uses for AOP2 and the corresponding gene, for example, development of reagents (antibodies, expression vectors, cell lines, congenic and transience mice) that may be used in the diagnosis and treatment of atherosclerosis and related disease states.

[0001] The government may own rights to this application or any patentissued thereto under the following grants: HL-32087 and DK-45639 fromthe National Institutes of Health.

BACKGROUND OF THE INVENTION

[0002] I. Field of the Invention

[0003] The present invention relates to the fields of medicine,molecular biology and biochemistry. More particular, the inventionrelates to the cloning, analysis and use of genes related toatherosclerotic disease, a clinically significant condition that maylead to heart attacks and strokes. Specifically, the invention relates,in one embodiment, to the novel gene and gene product, Aop2 and AOP2,respectively, and to the novel role of AOP2 as an antioxidant in theprotection against atherosclerotic disease.

[0004] II. Related Art

[0005] Atherosclerosis is a major contributing cause of heart disease,the leading cause of death in the United States. It is characterized bythe formation of elevated intimal fibrofatty plaques called atheromas,which narrow arterial lumens, damage the underlying media and are proneto undergo superimposed complications including calcification,ulceration with overlying thrombosis and intraplaque hemorrhage. Thecenters of these plaques often contain a lipid-rich debris containingnotably cholesterol and cholesterol esters.

[0006] Atherosclerosis has achieved considerable medical importancebecause of its predilection for the coronary arteries and the arterialsupply to the brain and the heart.

[0007] Atheromatous involvement of the coronary arteries underlies thedominant form of heart disease, i.e., coronary heart disease, also knownas ischemic heart disease, the most important form of which ismyocardial infarction. Ischemic heart disease represents about 90% ofall forms of heart disease, and myocardial infarction is the commonestcause of death in atherosclerosis-prone populations.

[0008] Atherosclerosis is virtually ubiquitous among the populations ofNorth America, Europe and the Soviet Union. In contrast, it is much lessprevalent in Central and South America, Africa, Asia and the Orient. Acomparison of Finland, which has the highest mortality rate fromischemic heart disease, with Japan shows a differential of overten-fold. The U.S. ranks in the top ten in mortality from ischemic heartdisease. Though it is clear that genetic predisposition exists, theprimary focus of diagnosis and treatment relates to environmentalinfluences, particularly smoking, sedentary lifestyles, obesity,hypertension, diet and plasma cholesterol levels.

[0009] It is well known that elevated levels of cholesterol in theplasma carry an increased risk of heart disease. However, thischolesterol is carried in both low density lipoproteins (LDL-C) and highdensity lipoproteins (HDL-C). It is the levels of LDL-C that carry theincreased risk to heart disease; elevated levels of HDL-C actuallyprotect from atherosclerosis and heart disease. To understand themechanism of how elevated LDL-C leads to the formation ofatherosclerosis in the arteries, one must turn to the earliest events inthe formation of the fatty streak, which is the precursor of theatherosclerotic plaque. The first step in the formation of a fattystreak is the accumulation of foam cells underneath the endotheliallayer of the artery. Foam cells are monocyte-derived macrophages filledwith fat, and these cells are the major cell type in fatty streaks. Thekey step that leads to the formation of foam cells is the oxidation ofLDL-C (reviewed by Steinberg et al., 1989; Netto et al., 1996; Witzum &Steinberg, 1991). Macrophages take up primarily oxidized LDL, so it isoxidized LDL that leads to the formation of foam cells and fattystreaks. Dietary antioxidants, such as vitamin E or natural antioxidantsin the body could protect LDL from oxidation. HDL-C also protects LDL-Cfrom oxidation (reviewed by Banka, 1996), but in the process HDL itselfbecomes oxidized. When oxidized, HDL carries most of the lipidperoxidation products in plasma (Bowry et al., 1992; Hahn et al., 1994)and its apoproteins are cross-linked by oxidative damage (Marcel et al.,1989). This damaged and oxidized HDL is cleared from the plasma morequickly than native HDL (Guertin et al., 1994; Senault et al, 1990). Theconsequences of HDL protection of LDL from oxidation results inrelatively high plasma HDL if very little oxidation of LDL is occuring,but if more oxidation is occurring, then HDL becomes oxidized, clearedfrom the plasma more rapidly, and plasma levels of HDL decrease.

[0010] Atherosclerosis is not unique to human populations. Athl is apreviously described gene locus that affects the concentration of highdensity lipoprotein cholesterol and susceptibility to atherosclerosisamong inbred strains of mice. The atherosclerosis-susceptible allele iscarried by strain C57BL/6J and the resistant alleles are carried bystrains C3H/HeJ and BALB/cJ. The susceptible phenotype of Athl ischaracterized by relatively low concentrations (35-45 mg/dl) of highdensity lipoprotein-cholesterol (HDL) in the plasma of female mice fed ahigh fat and high cholesterol diet and the development of fatty streaklesions at many places along the aorta (Paigen, 1985). These fattystreaks eventually develop into mature atherosclerotic plaques (Paigenet al., 1990). The resistant phenotype of Athl, found in strains BALB/cand C3H, is characterized by higher concentrations (60-90 mg/dl) ofplasma HDL and the absence of fatty streak lesions in the aorta whenmice are fed the atherogenic diet.

[0011] The Athl phenotype is caused by a single gene difference betweenstrains C57BL/6 and C3H and also between strains C57BL/6 and BALB/c.This conclusion was initially based on examination of recombinant inbred(RI) strains BXH and CXB (Paigen, 1987a) and confirmed by backcrossesbetween C57BL/6 and the two resistant strains (Paigen, 1987b, 1987c).Athl mapped to the distal end of mouse chromosome 1 at a distance of 6cM from the gene for apolipoprotein AII (Apoa2). Athl was clearlyseparate from Apoa2 as demonstrated by analysis of a congenic strain,B6.C-H25^(c)/By, which carries the apoAII gene from BALB/c but the Athlsusceptible gene from B6 (Paigen 1987a). Athl maps to the regionhomologous to chromosome 1q24-25 in humans.

[0012] Despite this useful information, there is very little directknowledge regarding the role of this gene in atherosclerotic disease.Not only is the identity of the gene unknown, its function remainspurely speculative. As a result, there is a considerable need in thefield for evidence that will establish a fictional link between Athl andheart disease, as well as information on the structural attributes ofthe gene product of Athl.

SUMMARY OF THE INVENTION

[0013] Therefore, it is a goal of the present invention to provideadditional information on the role of the Athl gene in atheroscleroticdisease. It is another goal of the present invention to providecompositions and methods for use in diagnosing and treating Athl relateddisease states. It also is a goal of the present invention to developreagents that can stimulate or inhibit functions related to Athl so toaddress pathologic states associated therewith.

[0014] In satisfying these objectives, there is provided an isolatedpolypeptide designated AOP2. The polypeptide may be from any species,preferably mammalian, and more preferably human or murine. In onespecific embodiment, the polypeptide has the sequence set forth in SEQID NO:2. In another specific embodiment, the polypeptide has thesequence set forth in SEQ ID NO:4.

[0015] In another embodiment, there is provided an antigen compositioncomprising an Aop2 polypeptide or a fragment thereof and apharmaceutically acceptable buffer or diluent. Again, the polypeptidemay be human or murine, and more specifically may be derived from thesequence of SEQ ID NO:2 or SEQ ID NO:4 respectively.

[0016] In still another embodiment, there is provided a nucleic acidencoding an AOP2 polypeptide. The nucleic acid may encode a humanpolypeptide or a murine polypeptide, among other mammalian andnon-mammalian polypeptides. The nucleic acid may encode a polypeptide ofSEQ ID NO:2 or SEQ ID NO:4 and, more specifically, the nucleic acid mayhave a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3.

[0017] In still yet another embodiment, there is provided anoligonucleotide comprising at least about 10 consecutive bases of thenucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3. Theoligonucleotide may be longer, for example, at least about 15, 20, 25,30, 35, 40, 45 or 50 consecutive bases of the nucleic acid set forth inSEQ ID NO:1 or SEQ ID NO:3.

[0018] In still yet a further embodiment, there is provided a method fordiagnosing a predisposition to atherosclerotic lesions in a subjectcomprising (i) obtaining a sample from said subject; and (ii) evaluatingsaid sample for the presence of an AOP2 polypeptide. The sample may beselected from the group consisting of heart, artery, vein, skin, muscle,facia, brain, prostate, breast, endometrium, lung, pancreas, smallintestine, blood cells, liver, testes, ovaries, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow or kidney, lymph fluid,ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid,lacrimal fluid, stool and urine. The subject may be a human.

[0019] The evaluating may comprise determining the antioxidant activityof an AOP2 polypeptide of said sample, determining the level of an AOP2polypeptide in cells of said sample or determining the sequence of anucleic acid from said sample that encodes an AOP2 polypeptide. Thedetermining may comprise quantitative PCR or contacting said sample withan antibody that binds immunologically to an AOP2 polypeptide.

[0020] In still yet a further embodiment, there is provided a method forscreening a compound for AOP2 stimulatory activity comprising (i)providing an AOP2 polypeptide having antioxidant activity; (ii)contacting said AOP2 polypeptide with a candidate stimulator; and (iii)determining the antioxidant activity of said AOP2 polypeptide in thepresence and absence of said candidate stimulator.

[0021] In yet another embodiment, there is provided a method forscreening a compound for antioxidant stimulatory activity comprising (i)providing a cell comprising an nucleic acid encoding an active AOP2polypeptide; (ii) contacting said cell with a candidate stimulator; and(iii) determining the antioxidant activity in said cell in the presenceand absence of said candidate stimulator. The cell may be located in anon-human animal.

[0022] In still yet another embodiment, there is provided a method forscreening a compound for anti-atherosclerotic activity comprising (i)providing a lipid; (ii) contacting said lipid with a candidateantioxidant; and (iii) determining the oxidation state of said lipid.

[0023] In still yet other embodiments, there are provided a monoclonalantibody that binds immunologically to an AOP2 polypeptide and apolyclonal antisera, antibodies of which bind immunologically to an AOP2polypeptide.

[0024] Yet further embodiments include an expression vector comprising anucleic acid encoding an AOP2 polypeptide, said nucleic acid positionedin operable relation to a promoter and a recombinant host cellcomprising a nucleic acid encoding an AOP2 polypeptide, said nucleicacid positioned in operable relation to a promoter.

[0025] In still a further embodiment, there is provided a method forincreasing AOP2 function in a cell comprising (i) providing a nucleicacid encoding an AOP2 polypeptide having antioxidant activity, saidnucleic acid positioned in operable relation to a promoter; and (ii)contacting said nucleic acid with said cell under conditions permittingthe uptake of said nucleic acid. The AOP2 polypeptide may be a humanpolypeptide, for example, as set forth in SEQ ID NO:2. The nucleic acidfurther may comprise an expression vector, and the expression vector maybe encapsulated in a liposome. The expression vector may be a viralvector, such as an adenoviral vector, a retroviral vector, a vacciniaviral vector, an adeno-associated viral vector or a herpesviral vector.The cell may be located in a human subject, in an experimental animal.The nucleic acid may be administered intravenously. The promoter may beCMV, RSV or E1A.

[0026] In another embodiment, there is provided a method of reducingatherosclerotic lesions in a subject comprising administering to saidsubject a compound with antioxidant composition. It is noted that anantioxidant may function to protect lipid directly or indeed break downfree radicals that could oxidize a lipid composition, thereby protectingthe lipid indirectly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

[0028]FIG. 1: LTW4 protein isoforms. Corresponding regions ofsilver-stained two-dimensional electrophoresis gels of liver homogenatesfrom C57BL/6 (top panel) and DBA/2 (bottom panel) are shown. The C57BL/6isoform of LTW4 has a MW of 26 kD and a pI of 5.9. The DBA/2 isoform ofLTW4 has a MW of 26 kD and a pI of 5.6.

[0029]FIG. 2: Consensus sequence for Aop2. Nucleotide 389 (shown inbold) is adenine in DBA/2J, C3H and BALB/c and cytosine in C57BL/6J. Thecodon containing this polymorphic nucleotide corresponds to asparticacid in DBA/2J and to alanine in C57BL/6J. The N-terminal amino acidsequence obtained by microsequence analysis is underlined.

[0030] FIGS. 3A & 3B: SSCP mapping analysis of Aop2. (FIG. 3A) Analysisof genomic DNA from parental strain controls and 22 progeny from the BSSinterspecific cross using an SSCP derived from 3′ UTR of Aop2. Sincethis is a (C57BL/6J X M. spretus) F1 X M. spretus backcross, all progenycarry at least one M. spretus allele. Heterozygotes also carrying aC57BL/6J allele are marked. B: C57BL/6J control; S: M spretus control;H: C57BL/6J -M. spretus heterozygote. (FIG. 3B) The position of Aop2 inthe BSS cross is shown on the left, and is compared to the position ofLtw4 on the chromosome 1 consensus map¹⁰ on the right. Interlocusdistances are shown in cM.Apoa2:apolipoprotein AII.

[0031]FIG. 4: Amino acid homologies of murine anti-oxidant proteins.Four members of the murine antioxidant protein family were analyzedusing the Blocks program (http://www.blocks.fhcrc.org¹⁸). Three domainsof conserved amino acids were identified and are shown. Amino acididentities are shown in bold. The two cysteines that are conserved inmost members of the TSA family are marked with an asterisk; the secondcysteine is not present in AOP2 or its human homologue.

[0032]FIG. 5: High resolution map of mouse chromosome 1. Polymorphicmarkers illustrated.

[0033]FIG. 6: (A) Genomic structure of the mouse Aop2 gene. Exons arerepresented by boxes; light portions indicated untranslated regions anddark portions indicate the coding region. The relative proportion ofexon and intron sizes are shown. Five exons and four introns spanapproximately 10.7 kb. (B) Sizes of exons and introns and splicejunction sequences.

[0034]FIG. 7: Nucleotide sequence of the 5′-flanking region of the mouseAop2 gene. Nucleotide position +1 corresponds to the 5′-terminus of themouse mGPx cDNA (Munz et al., J. Biochem. 326: 579-585 (1997)), andnegative numbers refer to 5′ flanking sequences. The translation startcodon ATG is underlined, and putative transcription factor binding sitesare boxed.

[0035]FIG. 8: (A) Alignment of the Aop2 nucleotide sequence with that ofthe Aop2-rs1 and Aop2-rs2. Reported sequences span the correspondingcoding region of Aop2. The arrow indicates the start of translation.Dashes represent nucleotide identities with Aop2; dots represent gaps inthe sequence. (B) Alignment of the Aop2 protein with the predicted aminoacid sequences encoded by Aop2-rs1 and Aop2-rs2. Dashes represent aminoacid identities with Aop2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. The PresentInvention

[0036] The present invention relates to the identification of a novelgene, designated Aop2, which maps to the previously describedatherosclerosis susceptibility locus, Athl. The Aop2 gene product, nowdesignated AOP2 is an antioxidant and belongs to a highly related familyof antioxidant proteins. The inventors have identified two differentalleles of Athl responsible for a difference in atherosclerosissusceptibility between mouse strains. Sequencing of Aop2 cDNA fromsusceptible and resistant mouse strains identified a single base pairdifference in the sequences of the two alleles. The inventors furtherhave determined the structure of the Aop2 gene. The Athl resistantallele, carried by inbred mouse strains C3H and BALB/c, must code for anAOP2 protein that functions well as an antioxidant while the Athlsusceptible allele, carried by strain C57BL/6, must code for an AOP2protein that functions less well as an antioxidant, permitting more LDLto be oxidized, more foam cells to form, more fatty streaks to raise,and more HDL-C to be damaged by oxidation products, thus resulting inmore rapid clearance of HDL and lower HDL levels in plasma. In otherembodiments, it has been demonstrated that phenotypic differences mayalso result from differences in Aop2 expression and not just proteinstructure. More specifically, data discussed in new Example 7 revealsdifferences in Aop2 mRNA levels. Example 8 discloses genomic sequenceinformation including potentially important regulatory elements in thepromoter region.

[0037] Using a high resolution mapping cross of over 1000 mice, theinventors were able to localize the atherosclerosis susceptibilitylocus, Athl, to narrow a genetic region on mouse chromosome 1, with norecombinants evident with the locus D1Mit424. The cross was betweenC57BL/6, a strain that is susceptible to atherosclerosis, and a congenicstrain constructed by several generations of backcrossing theatherosclerosis resistant strain Spretus to C57BL/6. The congenic straincarries mostly C57BL/6 genes except for a small region of Spretus geneson chromosome 1, extending at least from the markers D1Mit14 to D1Mit15.Heterozygous mice from a mating between C57BL/6 and the congenic strain,named C57BL/6.Sp-Athl^(r), were mated to C57BL/6. The resultingbackcross progeny were phenotyped for the size of their fatty streaklesions in the aorta and genotype for the loci in the relevant region ofmouse chromosome 1. Thus, Athl was narrowed to a very small region.

[0038] One gene that maps to this region is Ltw4, identified previouslyby variant polypeptides using two-dimensional gel electrophoresis(Elliott, 1979a; Elliott, 1979b; Elliott et al., 1980). This protein isexpressed in the liver and has a molecular weight of 20-30 kD. Theinventors used analytical and preparative two-dimensional gelelectrophoresis and identified variants of a 26 kD polypeptide whichdiffered in their isoelectric point between strains C57BL/6 and DBA/2 ina pattern similar to that originally described for LTW (Iakoubova etal., 1997). N-terminal amino acid sequence was obtained bymicrosequencing, and overlapping expressed sequence tags (ESTs)corresponding to a full-length coding sequence were identified.Confirmation of identity was made by mapping the gene usingsingle-strand conformational polymorphism (SSCP); the map positioncorresponded to that previously described for Ltw4. This gene then wasmapped in the same mice used for the high resolution mapping of Athl; norecombinants were found between this gene and Athl, strongly suggestingthat Athl and Ltw4 are the same.

[0039] The cDNA corresponding to Ltw4 was sequenced and found to havehomology to a class of proteins characterized as thiol-specificantioxidants that are protective against damage caused by oxidativestress (Chae et al., 1994). The murine MER5 gene also is a member ofthis gene family (Yamamoto et al., 1989) and recently has been renamedantioxidant protein 1, or Aop1, based on its functional characterization(Tsuji et al., 1995). Hence, the inventors propose that the Ltw4 gene bedesignated as antioxidant protein 2, or Aop2, and the correspondingprotein be designated as AOP2.

[0040] The cDNA for Aop2 was sequenced from atherosclerotic resistantand susceptible strains and a subset found to differ at a single base,nucleotide 389, that corresponds to an amino acid change in residue 124.This residue is the amino acid alanine in C57BL/6 mice, which aresusceptible to atherosclerosis, and aspartic acid in C3H mice, which areresistant to atherosclerosis. This amino acid change results in a changein isoelectric point that corresponds to the differences observed forLTW4.

[0041] Thus, the inventors propose that the difference inatherosclerosis susceptibility caused by Athl is determined by anantioxidant protein, AOP2, which differs between resistant andsusceptible strains. This proposal is based on the (i) concordance ofAop2 with Athl in a high resolution mapping cross, (ii) the isolationand sequencing of cDNA that differs between Athl susceptible and Athlresistant strains, (iii) the observation that the respective proteinproducts differ by an amino acid between the susceptible and resistantstrains, and (iv) the biological plausability of an antioxidant havingan important role in atherosclerosis, given that oxidation of plasmalipoproteins is of major importance in the atherosclerotic process.

[0042] The hypothesis that AOP2 differs in function between theresistant and susceptible strains further finds credibilty in the factthat oxidized LDL causes more foam cells to form, and hence more fattystreaks to develop. HDL protects LDL against oxidation, and when HDLbecomes damaged by oxidation products, it is cleared from the plasmamore readily. Significantly, the major differences between the Athlresistant and susceptible strains are in the size of the fatty lesionsand the levels of plasma HDL.

[0043] With the identification of the Aop2 gene as a contributing factorto atherosclerosis, a number of different uses become possible. Forexample, by screening for abnormalities in this gene, it now is possibleto ascertain, at the genetic or protein level, a predisposition toatherosclerotic disease. In addition, the association of this gene withantioxidant activity suggests the use of Athl mice strains to screen forcompounds that modulate antioxidant activity in vivo.

[0044] It also is possible to employ both protein and DNA compositionsin the treatment of oxidative damage, atherosclerosis and heart disease.With respect to the latter application, the identification of important,native, genomic regulatory elements, particularly those associated withincreased expression, is of paramount importance. The sequenceinformation presented in Example 8 enables the identification of suchcritical regulatory sequences. More specifically, FIG. 7, described inExample 8, describes several consensus recognition sequences for knowntranscription factors are found in the putative proximal promoter. Theseinclude potential binding sites for USF (upstream stimulatory factor)and SREBP (sterol response element binding protein). Both of these DNAbiding proteins have been demonstrated to be important in the regulationof gene expression. Although the particular promoter sequence describedis not associated with particularly high Aop2 transcription levels,given the identification of the consensus regulatory sequences, one ofskill in the art could isolate promoter sequences from allelescharacterized by increased levels of mRNA production and compare suchsequences with that disclosed in FIG. 7. Such analysis is likely toreveal the identity of important regulatory sequences responsible forthe differences in Aop2 expression documented herein.

[0045] The identification of such regulatory sequences can be exploitedtherapeutically in a gene therapy protocol. AOP2 activity appears to belocalized to the cytosol. A therapeutic approach for the treatment of anAthl susceptible individual is to introduce a mammalian expressionvector carrying an Aop2 allele conferring Athl resistance to therelevant cells of the individual. With respect to atherosclerosis, therelevant target cells would be the cells of the artery wall (the site oflow density lipoprotein (LDL) oxidation) Conventional mammalianexpression vectors are discussed in greater detail below. Previous workhas shown that the delivery of genes by such conventional mammalianvectors can result in expression in the cells of the artery wall. Inthis context, it is important to note that Aop2 is widely expressed inat least 23 distinct tissues. In light of this somewhat ubiquitousexpression pattern, deleterious effects associated with expression ofAop2 in cells other that the target cells would be highly unlikely.

II. AOP2 Polypeptides and Fragments Thereof

[0046] Thus, according to one aspect of the present invention, thepresent inventors provide the primary sequence for the polypeptide AOP2.This molecule will prove useful in a variety of different contexts. Forexample, AOP2 may be used as an antigen to raise antibodies that can, inturn, be used to study and diagnosis disease states relating to AOP2.AOP2 also can be used as part of screening assays to examine reagentsfor their ability to affect the function of AOP2 in vitro or in vivo.Finally, AOP2 may be used to prevent oxidative damage in vivo, forexample, during heart surgery where blood is oxygenated outside thebody.

[0047] In addition to the entire AOP2 molecule, the present inventionalso relates to fragments of the polypeptide that may or may not retainthe antioxidant (or other) activity. Fragments including the N-terminusof the molecule may be generated by genetic engineering of translationstop sites within the coding region (discussed below). Alternatively,treatment of the AOP2 molecule with proteolytic enzymes, known asprotease, can produces a variety of N-terminal, C-terminal and internalfragments. Examples of fragments may include contiguous residues of theAthl sequences given in SEQ ID NO:2 or SEQ ID NO:4, of 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400 or more aminoacids in length. These fragments may be purified according to knownmethods, such as precipitation (e.g., ammonium sulfate), HPLC, ionexchange chromatography, affinity chromatography (includingimmunoaffinity chromatography) or various size separations(sedimentation, gel electrophoresis, gel filtration).

A. Structural Features of the Polypeptide

[0048] The gene for Aop2 encodes a 224 amino acid polypeptide. Thepredicted molecular weight of this molecule is 26 kD. Thus, at aminimum, this molecule may be used as a standard in assays wheremolecule weight is being examined.

B. Functional Aspects

[0049] When the present application refers to the function of Aop2 or“wild-type” activity, it is meant that the molecule in question has theability to prevent oxidation of various biological molecules such aslipids, proteins and especially low and high density lipoproteins.Specifically, AOP2 is a member of a family of proteins that can protectenzymes from damage by thiol-dependent, metal catalyzed oxidation.Determination of which polypeptides possess this activity may bedetermined using assays familiar to those of skill in the art. Forexample, transfer of genes encoding Aop2, or variants thereof, intocells that do not have a functional Aop2 product, and hence exhibitexcess oxidation, will identify those molecules having AOP2 function.

[0050] For example, cell extracts may be assayed for protection ofenzymes from metal-catalyzed oxidation. There also is at least one yeaststrain deficient in AOP2-like function which may be used incomplementation studies with Aop2 genes. In a particular example, theprotein could be measured using an immunoassay with an antibody specificto that protein. The activity of this protein also could be measured byits ability to protect LDL from oxidation. Such assays are based on thequantity of oxidized LDL; if the antioxidant is functioning well, thequantity of oxidized LDL will be low and conversely if the antioxidanthas poor function, oxidized LDL will be in high quantity. Oxidized LDLcan be estimated directly by measuring the amount of thiobarbituric acidreactive substances (TBARS), with absorbance at 532 nm, that weregenerated (Ohkawa et al., 1979). This assay is used extensively inatherosclerosis research (Parthasarathy et al., 1990). Another method ofdirect estimation of oxidized LDL is to measure the products of lipidperoxidation, which are conjugated dienes and trienes, as described(Klimov et al., 1993). An indirect method of measuring oxidized LDL isuptake by macrophages. Since macrophages take up oxidized LDL but notnative LDL, the ability of macrophages to degrade¹²⁵I-labeled LDL isused (Parthasarathy et al., 1990). Finally, members of this class ofantioxidants can be assayed by their ability to protect glutaminesynthetase from oxidative inactivation by thiol-dependent metalcatalyzed oxidations systems (Netto et al., 1996). This involves removalof hydrogen peroxide from the oxidation system.

C. Variants of AOP2

[0051] Amino acid sequence variants of the polypeptide can besubstitutional, insertional or deletion variants. Deletion variants lackone or more residues of the native protein which are not essential forfunction or immunogenic activity. Insertional mutants typically involvethe addition of material at a non-terminal point in the polypeptide.This may include the insertion of an immunoreactive epitope or simply asingle residue. Terminal additions, called fusion proteins, arediscussed below.

[0052] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of this kind preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

[0053] The following is a discussion based upon changing of the aminoacids of a protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

[0054] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

[0055] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte &Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (-0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0056] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0057] It also is understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±1); glutamate(+3.0 ±1); serine (+0.3); asparagine (+0.2) glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5 ±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

[0058] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin +2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

[0059] As outlined above, amino acid substitutions are generally basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0060] Another embodiment for the preparation of polypeptides accordingto the invention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used, in conjunction with the principles outlineabove, to engineer second generation molecules having many of thenatural properties of AOP2, but with altered and even improvedcharacteristics.

D. Domain Switching

[0061] As described in the examples, the present inventors haveidentified putative murine and human homologues of the Aop2 gene. Inaddition, mutations have been identified in AOP2 (e.g., residue 122)which are believed to alter its function. These studies are importantfor at least two reasons. First, they provide a reasonable expectationthat still other homologues, allelic variants and mutants of this genemay exist in related species, such as dog, rat, rabbit, monkey, gibbon,chimp, ape, baboon, cow, pig, horse, sheep and cat. Upon isolation ofthese homologues, variants and mutants, and in conjunction with otheranalyses, certain active or functional domains can be identified.Second, this will provide a starting point for further mutationalanalysis of the molecule. One way in which this information can beexploited is in “domain switching.”

[0062] Domain switching involves the generation of chimeric moleculesusing different but, in this case, related polypeptides. By comparingthe mouse and human sequences for Aop2 with the Aop2 of other species,and with mutants and allelic variants of these polypeptides, one canmake predictions as to the functionally significant regions of thesemolecules. It is possible, then, to switch related domains of thesemolecules in an effort to determine the criticality of these regions toAOP2 function. These molecules may have additional value in that these“chimeras” can be distinguished from natural molecules, while possiblyproviding the same function.

[0063] Based on the sequence identity, at the amino acid level, of themouse, dog and human sequences, it may be inferred that even smallchanges in the primary sequence of the molecule will affect function.Further analysis of mutations and their predicted effect on secondarystructure will add to this understanding.

E. Fusion Proteins

[0064] A specialized kind of insertional variant is the fusion protein.This molecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

F. Purification of Proteins

[0065] It will be desirable to purify AOP2 or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

[0066] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an encoded protein or peptide. The term “purifiedprotein or peptide” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein orpeptide is purified to any degree relative to its naturally-obtainablestate. A purified protein or peptide therefore also refers to a proteinor peptide, free from the environment in which it may naturally occur.

[0067] Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

[0068] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS-PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0069] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0070] There is no general requirement that the protein or peptidealways be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

[0071] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS-PAGE (Capaldiet al., 1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

[0072] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0073] Gel chromatography, or molecular sieve chromatography, is aspecial type of partition chromatography that is based on molecularsize. The theory behind gel chromatography is that the column, which isprepared with tiny particles of an inert substance that contain smallpores, separates larger molecules from smaller molecules as they passthrough or around the pores, depending on their size. As long as thematerial of which the particles are made does not adsorb the molecules,the sole factor determining rate of flow is the size. Hence, moleculesare eluted from the column in decreasing size, so long as the shape isrelatively constant. Gel chromatography is unsurpassed for separatingmolecules of different size because separation is independent of allother factors such as pH, ionic strength, temperature, etc. There alsois virtually no adsorption, less zone spreading and the elution volumeis related in a simple matter to molecular weight.

[0074] Affinity Chromatography is a chromatographic procedure thatrelies on the specific affinity between a substance to be isolated and amolecule that it can specifically bind to. This is a receptor-ligandtype interaction. The column material is synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial is then able to specifically adsorb the substance from thesolution. Elution occurs by changing the conditions to those in whichbinding will not occur (alter pH, ionic strength, temperature, etc.).

[0075] A particular type of affinity chromatography useful in thepurification of carbohydrate containing compounds is lectin affinitychromatography. Lectins are a class of substances that bind to a varietyof polysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

[0076] The matrix should be a substance that itself does not adsorbmolecules to any significant extent and that has a broad range ofchemical, physical and thermal stability. The ligand should be coupledin such a way as to not affect its binding properties. The ligand shouldalso provide relatively tight binding. And it should be possible toelute the substance without destroying the sample or the ligand. One ofthe most common forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

G. Synthetic Peptides

[0077] The present invention also describes smaller AOP2-relatedpeptides for use in various embodiments of the present invention.Because of their relatively small size, the peptides of the inventioncan also be synthesized in solution or on a solid support in accordancewith conventional techniques. Various automatic synthesizers arecommercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young, (1984); Tam et al.,(1983); Merrifield, (1986); and Barany and Merrifield (1979), eachincorporated herein by reference. Short peptide sequences, or librariesof overlapping peptides, usually from about 6 up to about 35 to 50 aminoacids, which correspond to the selected regions described herein, can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides. Alternatively, recombinant DNA technologymay be employed wherein a nucleotide sequence which encodes a peptide ofthe invention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

H. Antigen Compositions

[0078] The present invention also provides for the use of AOP2 proteinsor peptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that either AOP2, or portionsthereof, will be coupled, bonded, bound, conjugated or chemically-linkedto one or more agents via linkers, polylinkers or derivatized aminoacids. This may be performed such that a bispecific or multivalentcomposition or vaccine is produced. It is further envisioned that themethods used in the preparation of these compositions will be familiarto those of skill in the art and should be suitable for administrationto animals, i.e., pharmaceutically acceptable. Preferred agents are thecarriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin(BSA).

III. Nucleic Acids

[0079] The present invention also provides, in another embodiment, genesencoding AOP2. Genes for the human and murine AOP2 molecule have beenidentified. The present invention is not limited in scope to thesegenes, however, as one of ordinary skill in the art could, using thesetwo nucleic acids, readily identify related homologues in various otherspecies (e.g., dog, rat, rabbit, monkey, gibbon, chimp, ape, baboon,cow, pig, horse, sheep, cat and other species). The finding of a mousehomologue for this gene makes it likely that other species more closelyrelated to humans will, in fact, have a homologue as well. These genemay be used as part of diagnostic and therapeutic regimens.

[0080] In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, “an Aop2 gene” may contain a variety of different bases and yetstill produce a corresponding polypeptides that is functionallyindistinguishable, and in some cases structurally, from the human andmouse genes disclosed herein.

[0081] Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the function of Aop2.

A. Nucleic Acids Encoding Aop2

[0082] The human gene disclosed in SEQ ID NO: 1 and the murine genedisclosed in SEQ ID NO:3 are Aop2 genes of the present invention.Nucleic acids according to the present invention may encode an entireAOP2 product, a domain of AOP2, or any other fragment of AOP2 sequencesset forth herein. The nucleic acid may be derived from genomic DNA,i.e., cloned directly from the genome of a particular organism. Inpreferred embodiments, however, the nucleic acid would comprisecomplementary DNA (cDNA). Also contemplated is a cDNA plus a naturalintron or an intron derived from another gene; such engineered moleculesare sometime referred to as “mini-genes.” At a minimum, these and othernucleic acids of the present invention may be used as molecular weightstandards in, for example, gel electrophoresis.

[0083] The term “cDNA” is intended to refer to DNA prepared usingmessenger RNA (mRNA) as template. The advantage of using a cDNA, asopposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There may be times whenthe full or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

[0084] It also is contemplated that a given Aop2 gene from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 below).

[0085] As used in this application, the term “a nucleic acid encoding anAop2” refers to a nucleic acid molecule that has been isolated free oftotal cellular nucleic acid. In preferred embodiments, the inventionconcerns a nucleic acid sequence essentially as set forth in SEQ ID NO:1or SEQ ID NO:3. The term “as set forth in SEQ ID NO:1 or SEQ ID NO:3”means that the nucleic acid sequence substantially corresponds to aportion of SEQ ID NO:1 or SEQ ID NO:3. The term “functionally equivalentcodon” is used herein to refer to codons that encode the same aminoacid, such as the six codons for arginine or serine (Table 1, below),and also refers to codons that encode biologically equivalent aminoacids, as discussed in the following pages. TABLE 1 Amino Acids CodonsAlanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp DGAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GlycineGly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUCAUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0086] Allowing for the degeneracy of the genetic code, sequences thathave at least about 50%, usually at least about 60%, more usually about70%, most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NO: 1 or SEQ ID NO:3 will be sequences that are“as set forth in SEQ ID NO:1 or SEQ ID NO:3.” Sequences that areessentially the same as those set forth in FIG. X may also befunctionally defined as sequences that are capable of hybridizing to anucleic acid segment containing the complement of SEQ ID NO: 1 or SEQ IDNO:3 under standard conditions.

[0087] The DNA segments of the present invention include those encodingbiologically functional equivalent AOP2 proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers

[0088] Naturally, the present invention also encompasses DNA segmentsthat are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO: 1 or SEQ ID NO:3. Nucleic acid sequences thatare “complementary” are those that are capable of base-pairing accordingto the standard Watson-Crick complementary rules. As used herein, theterm “complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 or SEQ ID NO:3under relatively stringent conditions such as those described herein.Such sequences may encode the entire Aop2 protein or functional ornon-functional fragments thereof.

[0089] Alternatively, the hybridizing segments may be shorteroligonucleotides. Sequences of 17 bases long should occur only once inthe human genome and, therefore, suffice to specify a unique targetsequence. Although shorter oligomers are easier to make and increase invivo accessibility, numerous other factors are involved in determiningthe specificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or3431 bases and longer are contemplated as well. Such oligonucleotideswill find use, for example, as probes in Southern and Northern blots andas primers in amplification reactions.

[0090] Suitable hybridization conditions will be well known to those ofskill in the art. In certain applications, for example, substitution ofamino acids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

[0091] In other embodiments, hybridization may be achieved underconditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl₂, 10 mM dithiothreitol, at temperatures between approximately 20°C. to about 37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

[0092] One method of using probes and primers of the present inventionis in the search for genes related to Aop2 or, more particularly,homologues of Aop2 from other species. The existence of a murinehomologue strongly suggests that other homologues of the human Aop2 willbe discovered in species more closely related, and perhaps more remote,than mouse. Normally, the target DNA will be a genomic or cDNA library,although screening may involve analysis of RNA molecules. By varying thestringency of hybridization, and the region of the probe, differentdegrees of homology may be discovered.

[0093] Another way of exploiting probes and primers of the presentinvention is in site-directed, or site-specific mutagenesis.Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0094] The technique typically employs a bacteriophage vector thatexists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage vectors are commercially available and their useis generally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

[0095] In general, site-directed mutagenesis is performed by firstobtaining a single-stranded vector, or melting of two strands of adouble-stranded vector which includes within its sequence a DNA sequenceencoding the desired protein. An oligonucleotide primer bearing thedesired mutated sequence is synthetically prepared. This primer is thenannealed with the single-stranded DNA preparation, taking into accountthe degree of mismatch when selecting hybridization conditions, andsubjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

[0096] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Antisense Constructs

[0097] In some cases, mutant proteins may not be non-functional. Rather,they may have aberrant functions that cannot be overcome by replacementgene therapy, even where the “wild-type” molecule is expressed inamounts in excess of the mutant polypeptide. Antisense treatments areone way of addressing this situation. Antisense technology also may beused to “knock-out” function of AOP2 in the development of cell lines ortransience mice for research, diagnostic and screening purposes.

[0098] Antisense methodology takes advantage of the fact that nucleicacids tend to pair with “complementary” sequences. By complementary, itis meant that polynucleotides are those which are capable ofbase-pairing according to the standard Watson-Crick complementarityrules. That is, the larger purines will base pair with the smallerpyrimidines to form combinations of guanine paired with cytosine (G:C)and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. Inclusion of lesscommon bases such as inosine, 5-methylcytosine, 6-methyladenine,hypoxanthine and others in hybridizing sequences does not interfere withpairing.

[0099] Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

[0100] Antisense constructs may be designed to bind to the promoter andother control regions, exons, introns or even exon-intron boundaries ofa gene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

[0101] As stated above, “complementary” or “antisense” meanspolynucleotide sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, sequencesof fifteen bases in length may be termed complementary when they havecomplementary nucleotides at thirteen or fourteen positions. Naturally,sequences which are completely complementary will be sequences which areentirely complementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

[0102] It may be advantageous to combine portions of genomic DNA withcDNA or synthetic sequences to generate specific constructs. Forexample, where an intron is desired in the ultimate construct, a genomicclone will need to be used. The cDNA or a synthesized polynucleotide mayprovide more convenient restriction sites for the remaining portion ofthe construct and, therefore, would be used for the rest of thesequence.

D. Ribozymes

[0103] Another approach for addressing the “dominant negative” mutantsis through the use of ribozymes. Although proteins traditionally havebeen used for catalysis of nucleic acids, another class ofmacromolecules has emerged as useful in this endeavor. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forsterand Symons, 1987). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurekand Shub, 1992). This specificity has been attributed to the requirementthat the substrate bind via specific base-pairing interactions to theinternal guide sequence (“IGS”) of the ribozyme prior to chemicalreaction.

[0104] Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

[0105] Within certain embodiments expression vectors are employed toexpress the Aop2 polypeptide product, which can then be purified and,for example, be used to vaccinate animals to generate antisera ormonoclonal antibody with which further studies may be conducted. Inother embodiments, the expression vectors are used in gene therapy.Expression requires that appropriate signals be provided in the vectors,and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

(i) Regulatory Elements

[0106] Throughout this application, the term “expression construct” ismeant to include any type of genetic construct containing a nucleic acidcoding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

[0107] In one embodiment, the expression construct will compriseregulatory sequences found, in nature, in operable relation to the Aop2gene. In certain embodiments, these regulatory sequences are fused to areporter or indicator gene, such as luciferase or lacZ. In otherembodiments, these regulatory sequences are operably connected to theAop2 gene, either as they would be found in nature or in a modifiedfashion.

[0108] In preferred embodiments, the nucleic acid encoding a geneproduct is under transcriptional control of a promoter. A “promoter”refers to a DNA sequence recognized by the synthetic machinery of thecell, or introduced synthetic machinery, required to initiate thespecific transcription of a gene. The phrase “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

[0109] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0110] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0111] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either cooperatively or independentlyto activate transcription.

[0112] The particular promoter employed to control the expression of anucleic acid sequence of interest is not believed to be important, solong as it is capable of directing the expression of the nucleic acid inthe targeted cell. Thus, where a human cell is targeted, it ispreferable to position the nucleic acid coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter.

[0113] In various embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter, the Rous sarcoma viruslong terminal repeat, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose.

[0114] By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalelements/promoters which may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

[0115] Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0116] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Promoters and enhancers are often overlapping andcontiguous, often seeming to have a very similar modular organization.

[0117] Below is a list of viral promoters, cellular promoters/enhancersand inducible promoters/enhancers that could be used in combination withthe nucleic acid encoding a gene of interest in an expression construct(Table 2 and Table 3). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct. TABLE 2ENHANCER/PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globine-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α1-Antitrypsin H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV4O Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

[0118] TABLE 3 Element Inducer MT II Phorbol Ester (TPA) Heavy metalsMMTV (mouse mammary tumor Glucocorticoids virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV4O PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP7O Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid HormoneGene Insulin E Box Glucose

[0119] Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

(ii) Selectable Markers

[0120] In certain embodiments of the invention, the cells containnucleic acid constructs of the present invention, a cell may beidentified in vitro or in vivo by including a marker in the expressionconstruct. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants, for example, genes thatconfer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocinand histidinol are useful selectable markers. Alternatively, enzymessuch as herpes simplex virus thymidine kinase (tk) or chloramphenicolacetyltransferase (CAT) may be employed. Immunologic markers also can beemployed. The selectable marker employed is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable markers are well known to one of skill in the art.

(iii) Multigene Constructs and IRES

[0121] In certain embodiments of the invention, the use of internalribosome binding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picanovirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

[0122] Any heterologous open reading frame can be linked to IRESelements. This includes genes for secreted proteins, multi-subunitproteins, encoded by independent genes, intracellular or membrane-boundproteins and selectable markers. In this way, expression of severalproteins can be simultaneously engineered into a cell with a singleconstruct and a single selectable marker.

(iv) Delivery of Expression Vectors

[0123] There are a number of ways in which expression vectors may beintroduced into cells. In certain embodiments of the invention, theexpression construct comprises a virus or engineered construct derivedfrom a viral genome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

[0124] One of the preferred methods for in vivo delivery involves theuse of an adenovirus expression vector. “Adenovirus expression vector”is meant to include those constructs containing adenovirus sequencessufficient to (a) support packaging of the construct and (b) to expressan antisense polynucleotide that has been cloned therein. In thiscontext, expression does not require that the gene product besynthesized.

[0125] The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

[0126] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0127] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0128] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the E1 , the D3 or bothregions (Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1 -deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

[0129] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

[0130] Recently, Racher et al. (1995) disclosed improved methods forculturing 293 cells and propagating adenovirus. In one format, naturalcell aggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

[0131] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0132] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

[0133] Adenovirus is easy to grow and manipulate and exhibits broad hostrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

[0134] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animalstudies suggested that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet and Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz and Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

[0135] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0136] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol and env genes but without the LTR andpackaging components is constructed (Mann et al, 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

[0137] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of lactoseresidues to the viral envelope. This modification could permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

[0138] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

[0139] There are certain limitations to the use of retrovirus vectors inall aspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

[0140] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

[0141] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

[0142] In order to effect expression of sense or antisense geneconstructs, the expression construct must be delivered into a cell. Thisdelivery may be accomplished in vitro, as in laboratory procedures fortransforming cells lines, or in vivo or ex vivo, as in the treatment ofcertain disease states. One mechanism for delivery is via viralinfection where the expression construct is encapsidated in aninfectious viral particle.

[0143] Several non-viral methods for the transfer of expressionconstructs into cultured mammalian cells also are contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

[0144] Once the expression construct has been delivered into the cellthe nucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

[0145] In yet another embodiment of the invention, the expressionconstruct may simply consist of naked recombinant DNA or plasmids.Transfer of the construct may be performed by any of the methodsmentioned above which physically or chemically permeabilize the cellmembrane. This is particularly applicable for transfer in vitro but itmay be applied to in vivo use as well. Dubensky et al (1984)successfully injected polyomavirus DNA in the form of calcium phosphateprecipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection. Benvenistyand Neshif (1986) also demonstrated that direct intraperitonealinjection of calcium phosphate-precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest may also be transferred in a similar manner in vivoand express the gene product.

[0146] In still another embodiment of the invention for transferring anaked DNA expression construct into cells may involve particlebombardment. This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

[0147] Selected organs including the liver, skin, and muscle tissue ofrats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin etal, 1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

[0148] In a further embodiment of the invention, the expressionconstruct may be entrapped in a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

[0149] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Wong et al., (1980) demonstratedthe feasibility of liposome-mediated delivery and expression of foreignDNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

[0150] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

[0151] Other expression constructs which can be employed to deliver anucleic acid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

[0152] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

[0153] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

[0154] In certain embodiments, gene transfer may more easily beperformed under ex vivo conditions. Ex vivo gene therapy refers to theisolation of cells from an animal, the delivery of a nucleic acid intothe cells in vitro, and then the return of the modified cells back intoan animal. This may involve the surgical removal of tissue/organs froman animal or the primary culture of cells and tissues.

[0155] Primary mammalian cell cultures may be prepared in various ways.In order for the cells to be kept viable while in vitro and in contactwith the expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

[0156] One embodiment of the foregoing involves the use of gene transferto immortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

[0157] Examples of useful mammalian host cell lines are Vero and HeLacells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293,HepG2, NIH3T3, RIN and

[0158] MDCK cells. In addition, a host cell strain may be chosen thatmodulates the expression of the inserted sequences, or modifies andprocesses the gene product in the manner desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

[0159] A number of selection systems may be used including, but notlimited to, HSV thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosphoribosyltransferase genes,in tk-, hgprt- or aprt- cells, respectively. Also, anti-metaboliteresistance can be used as the basis of selection for dhfr, that confersresistance to; gpt, that confers resistance to mycophenolic acid; neo,that confers resistance to the aminoglycoside G418; and hygro, thatconfers resistance to hygromycin.

[0160] Animal cells can be propagated in vitro in two modes: asnon-anchorage dependent cells growing in suspension throughout the bulkof the culture or as anchorage-dependent cells requiring attachment to asolid substrate for their propagation (i.e., a monolayer type of cellgrowth).

[0161] Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

[0162] Large scale suspension culture of mammalian cells in stirredtanks is a common method for production of recombinant proteins. Twosuspension culture reactor designs are in wide use—the stirred reactorand the airlift reactor. The stirred design has successfully been usedon an 8000 liter capacity for the production of interferon. Cells aregrown in a stainless steel tank with a height-to-diameter ratio of 1:1to 3:1. The culture is usually mixed with one or more agitators, basedon bladed disks or marine propeller patterns. Agitator systems offeringless shear forces than blades have been described. Agitation may bedriven either directly or indirectly by magnetically coupled drives.Indirect drives reduce the risk of microbial contamination through sealson stirrer shafts.

[0163] The airlift reactor, also initially described for microbialfermentation and later adapted for mammalian culture, relies on a gasstream to both mix and oxygenate the culture. The gas stream enters ariser section of the reactor and drives circulation. Gas disengages atthe culture surface, causing denser liquid free of gas bubbles to traveldownward in the downcomer section of the reactor. The main advantage ofthis design is the simplicity and lack of need for mechanical mixing.Typically, the height-to-diameter ratio is 10:1. The airlift reactorscales up relatively easily, has good mass transfer of gases andgenerates relatively low shear forces.

[0164] The antibodies of the present invention are particularly usefulfor the isolation of antigens by immunoprecipitation.Immunoprecipitation involves the separation of the target antigencomponent from a complex mixture, and is used to discriminate or isolateminute amounts of protein. For the isolation of membrane proteins cellsmust be solubilized into detergent micelles. Nonionic salts arepreferred, since other agents such as bile salts, precipitate at acid pHor in the presence of bivalent cations. Antibodies are and their usesare discussed further, below.

IV. Generating Antibodies Reactive With AOP2

[0165] In another aspect, the present invention contemplates an antibodythat is immunoreactive with a AOP2 molecule of the present invention, orany portion thereof. An antibody can be a polyclonal or a monoclonalantibody. In a preferred embodiment, an antibody is a monoclonalantibody. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Howell and Lane, 1988).

[0166] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide of the presentinvention and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a non-humananimal including rabbits, mice, rats, hamsters, pigs or horses. Becauseof the relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

[0167] Antibodies, both polyclonal and monoclonal, specific for isoformsof antigen may be prepared using conventional immunization techniques,as will be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

[0168] It is proposed that the monoclonal antibodies of the presentinvention will find useful application in standard immunochemicalprocedures, such as ELISA and Western blot methods and inimmunohistochemical procedures such as tissue staining, as well as inother procedures which may utilize antibodies specific to AOP2-relatedantigen epitopes. Additionally, it is proposed that monoclonalantibodies specific to the particular AOP2 of different species may beutilized in other useful applications In general, both polyclonal andmonoclonal antibodies against AOP2 may be used in a variety ofembodiments. For example, they may be employed in antibody cloningprotocols to obtain cDNAs or genes encoding other AOP2. They may also beused in inhibition studies to analyze the effects of AOP2-relatedpeptides in cells or animals. Anti-AOP2 antibodies will also be usefulin immunolocalization studies to analyze the distribution of AOP2 duringvarious cellular events, for example, to determine the cellular ortissue-specific distribution of AOP2 polypeptides under different pointsin the cell cycle. A particularly useful application of such antibodiesis in purifying native or recombinant AOP2, for example, using anantibody affinity column. The operation of all such immunologicaltechniques will be known to those of skill in the art in light of thepresent disclosure.

[0169] Means for preparing and characterizing antibodies are well knownin the art (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

[0170] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0171] As also is well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0172] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster, injection mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs.

[0173] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified AOP2 protein, polypeptide orpeptide or cell expressing high levels of AOP2. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, 1986), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0174] Following immunization, somatic cells with the potential forproducing antibodies, specifically B-lymphocytes (B-cells), are selectedfor use in the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0175] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0176] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, 1986; Campbell, 1984). For example,where the immunized animal is a mouse, one may use P3-X63/Ag8,P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with cell fusions.

[0177] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 ratio, though the ratio may vary from about20:1 to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

[0178] Fusion procedures usually produce viable hybrids at lowfrequencies, around 1×10⁻⁶ to 1×10^(−8.) However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0179] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B-cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B-cells.

[0180] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0181] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

V. Diagnosing Disease States Involving AOP2

[0182] The present inventors have determined that alterations in AOP2and the related gene are associated with atherosclerosis. Therefore,AOP2 and the corresponding gene may be employed as a diagnostic orprognostic indicator of atherosclerosis and related disease states. Morespecifically, point mutations, deletions, insertions or regulatorypertubations relating to AOP2 may cause or promote atherosclerosis orrelated diseases.

A. Genetic Diagnosis

[0183] One embodiment of the present invention comprises a method fordetecting variation in the expression of AOP2. This may comprisedetermining the level of AOP2 expressed in cells or determining specificalterations in the expressed product. Obviously, this sort of assay hasimportance in the diagnosis of atherosclerosis and related diseases.Related disease states include coronary heart disease, ischemic heartdisease and stroke.

[0184] The biological sample can be any tissue or fluid. Variousembodiments include cells of the heart, blood circulatory system(arteries, veins endothelial lining of any vessel), skin, muscle, facia,brain, prostate, breast, endometrium, lung, head & neck, pancreas, smallintestine, blood cells, liver, testes, ovaries, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney or immune cells(e.g., monocytes, macrophages). Other embodiments include fluid samplessuch as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.

[0185] Nucleic acid used is isolated from cells contained in thebiological sample, according to standard methodologies (Sambrook et al.,1989). The nucleic acid may be genomic DNA or fractionated or whole cellRNA. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA. Normally, the nucleic acid is amplified.

[0186] Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

[0187] Following detection, one may compare the results seen in a givenpatient with a statistically significant reference group of normalpatients and patients that have AOP2-related pathologies. In this way,it is possible to correlate the amount or kind of AOP2 detected withvarious clinical states or predisposition to clinical states.

[0188] Various types of defects are to be identified. Thus,“alterations” should be read as including deletions, insertions, pointmutations and duplications. Point mutations result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose occurring in non-germline tissues. Germ-line tissue can occur inany tissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of AOP2 produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

[0189] A variety of different assays are contemplated in this regard,including but not limited to, fluorescent in situ hybridization (FISH),direct DNA sequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR-SSCP.

(i) Primers and Probes

[0190] The term primer, as defined herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty base pairs in length, but longersequences can be employed. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.Probes are defined differently, although they may act as primers.Probes, while perhaps capable of priming, are designed to binding to thetarget DNA or RNA and need not be used in an amplification process.

[0191] In preferred embodiments, the probes or primers are labeled withradioactive species (³²P, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

(ii) Template Dependent Amplification Methods

[0192] A number of template dependent processes are available to amplifythe marker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

[0193] Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

[0194] A reverse transcriptase PCR amplification procedure may beperformed in order to quantify the amount of mRNA amplified. Methods ofreverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable, RNA-dependent DNA polymerases. These methods aredescribed in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

[0195] Another method for amplification is the ligase chain reaction (”LCR“), disclosed in EPO No. 320 308, incorporated herein by reference inits entirety. In LCR, two complementary probe pairs are prepared, and inthe presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

[0196] Qbeta Replicase, described in PCT Application No. PCT/US87/00880,may also be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

[0197] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[alpha-thio]-triphosphatesin one strand of a restriction site may also be useful in theamplification of nucleic acids in the present invention, Walker et al.(1992).

[0198] Strand Displacement Amplification (SDA) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis, i.e., nicktranslation. A similar method, called Repair Chain Reaction (RCR),involves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.Target specific sequences can also be detected using a cyclic probereaction (CPR). In CPR, a probe having 3′ and 5′ sequences ofnon-specific DNA and a middle sequence of specific RNA is hybridized toDNA that is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products that are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

[0199] Still another amplification methods described in GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

[0200] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR (Kwoh et al., 1989;Gingeras et al., PCT Application WO 88/10315, incorporated herein byreference in their entirety). In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double-stranded DNAmolecules are heat denatured again. In either case the single-strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7 or SP6. Inan isothermal cyclic reaction, the RNA's are reverse transcribed intosingle-stranded DNA, which is then converted to double-stranded DNA, andthen transcribed once again with an RNA polymerase such as T7 or SP6.The resulting products, whether truncated or complete, indicate targetspecific sequences.

[0201] Davey et al., EPO No. 329 822 (incorporated herein by referencein its entirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow”fragment ofE. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”)molecule, having a sequence identical to that of the original RNAbetween the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0202] Miller et al., PCT Application WO 89/06700 (incorporated hereinby reference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA” ) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, M. A., In: PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS, Academic Press, N.Y., 1990; Ohara et al., 1989; eachherein incorporated by reference in their entirety).

[0203] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

(iii) Southern/Northern Blotting

[0204] Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting of RNA species.

[0205] Briefly, a probe is used to target a DNA or RNA species that hasbeen immobilized on a suitable matrix, often a filter of nitrocellulose.The different species should be spatially separated to facilitateanalysis. This often is accomplished by gel electrophoresis of nucleicacid species followed by “blotting” on to the filter.

[0206] Subsequently, the blotted target is incubated with a probe(usually labeled) under conditions that promote denaturation andrehybridization. Because the probe is designed to base pair with thetarget, the probe will binding a portion of the target sequence underrenaturing conditions. Unbound probe is then removed, and detection isaccomplished as described above.

(iv) Separation Methods

[0207] It normally is desirable, at one stage or another, to separatethe amplification product from the template and the excess primer forthe purpose of determining whether specific amplification has occurred.In one embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

[0208] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography (Freifelder,1982).

(v) Detection Methods

[0209] Products may be visualized in order to confirm amplification ofthe marker sequences. One typical visualization method involves stainingof a gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

[0210] In one embodiment, visualization is achieved indirectly.Following separation of amplification products, a labeled nucleic acidprobe is brought into contact with the amplified marker sequence. Theprobe preferably is conjugated to a chromophore but may be radiolabeled.In another embodiment, the probe is conjugated to a binding partner,such as an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

[0211] In one embodiment, detection is by a labeled probe. Thetechniques involved are well known to those of skill in the art and canbe found in many standard books on molecular protocols. See Sambrook etal., 1989. For example, chromophore or radiolabel probes or primersidentify the target during or following amplification.

[0212] One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

[0213] In addition, the amplification products described above may besubjected to sequence analysis to identify specific kinds of variationsusing standard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the Aop2 gene that may then be analyzed by directsequencing.

(vi) Kit Components

[0214] All the essential materials and reagents required for detectingand sequencing Aop2 and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™ etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

(vii) Design and Theoretical Considerations for Relative QuantitativeRT-PCR

[0215] Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR (RT-PCR) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

[0216] In PCR, the number of molecules of the amplified target DNAincrease by a factor approaching two with every cycle of the reactionuntil some reagent becomes limiting. Thereafter, the rate ofamplification becomes increasingly diminished until there is no increasein the amplified target between cycles. If a graph is plotted in whichthe cycle number is on the X axis and the log of the concentration ofthe amplified target DNA is on the Y axis, a curved line ofcharacteristic shape is formed by connecting the plotted points.Beginning with the first cycle, the slope of the line is positive andconstant. This is said to be the linear portion of the curve. After areagent becomes limiting, the slope of the line begins to decrease andeventually becomes zero. At this point the concentration of theamplified target DNA becomes asymptotic to some fixed value. This issaid to be the plateau portion of the curve.

[0217] The concentration of the target DNA in the linear portion of thePCR amplification is directly proportional to the starting concentrationof the target before the reaction began. By determining theconcentration of the amplified products of the target DNA in PCRreactions that have completed the same number of cycles and are in theirlinear ranges, it is possible to determine the relative concentrationsof the specific target sequence in the original DNA mixture. If the DNAmixtures are cDNAs synthesized from RNAs isolated from different tissuesor cells, the relative abundances of the specific mRNA from which thetarget sequence was derived can be determined for the respective tissuesor cells. This direct proportionality between the concentration of thePCR products and the relative mRNA abundances is only true in the linearrange of the PCR reaction.

[0218] The final concentration of the target DNA in the plateau portionof the curve is determined by the availability of reagents in thereaction mix and is independent of the original concentration of targetDNA. Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR for acollection of RNA populations is that the concentrations of theamplified PCR products must be sampled when the PCR reactions are in thelinear portion of their curves.

[0219] The second condition that must be met for an RT-PCR experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCRexperiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for B-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

[0220] Most protocols for competitive PCR utilize internal PCR standardsthat are approximately as abundant as the target. These strategies areeffective if the products of the PCR amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

[0221] The above discussion describes theoretical considerations for anRT-PCR assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR is performed as a relative quantitative RT-PCRwith an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

[0222] Other studies may be performed using a more conventional relativequantitative RT-PCR assay with an external standard protocol. Theseassays sample the PCR products in the linear portion of theiramplification curves. The number of PCR cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR assays can be superior to those derived from the relativequantitative RT-PCR assay with an internal standard.

[0223] One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(viii) Chip Technologies

[0224] Specifically contemplated by the present inventors are chip-basedDNA technologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al. (1994); Fodor et al. (1991).

B. Immunodiagnosis

[0225] Antibodies of the present invention can be used in characterizingthe AOP2 content of healthy and diseased tissues, through techniquessuch as ELISAs and Western blotting. This may provide a screen for thepresence or absence of antioxidant activity or as a predictor ofatherosclerosis.

[0226] The use of antibodies of the present invention, in an ELISA assayis contemplated. For example, anti-AOP2 antibodies are immobilized ontoa selected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

[0227] After binding of antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with the sampleto be tested in a manner conducive to immune complex (antigen/antibody)formation.

[0228] Following formation of specific immunocomplexes between the testsample and the bound antibody, and subsequent washing, the occurrenceand even amount of immunocomplex formation may be determined bysubjecting same to a second antibody having specificity for AOP2 thatdiffers from the first antibody. Appropriate conditions preferablyinclude diluting the sample with diluents such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween®. These addedagents also tend to assist in the reduction of nonspecific background.The layered antisera is then allowed to incubate for from about 2 toabout 4 hr, at temperatures preferably on the order of about 25° toabout 27° C. Following incubation, the antisera-contacted surface iswashed so as to remove non-immunocomplexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween®, or boratebuffer.

[0229] To provide a detecting means, the second antibody will preferablyhave an associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

[0230] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonicacid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label.Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectrum spectrophotometer.

[0231] The preceding format may be altered by first binding the sampleto the assay plate. Then, primary antibody is incubated with the assayplate, followed by detecting of bound primary antibody using a labeledsecond antibody with specificity for the primary antibody.

[0232] The antibody compositions of the present invention will findgreat use in immunoblot or Western blot analysis. The antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. Immunologically-based detection methods for use inconjunction with Western blotting include enzymatically-, radiolabel-,or fluorescently-tagged secondary antibodies against the toxin moietyare considered to be of particular use in this regard.

VI. Methods for Screening Active Compounds

[0233] The present invention also contemplates the use of AOP2 andactive fragments, and nucleic acids coding therefor, in the screening ofcompounds for activity in either stimulating AOP2 activity, overcomingthe lack of AOP2 or blocking the negative effects of a mutant AOP2molecule. These assays may make use of a variety of different formatsand may depend on the kind of “activity” for which the screen is beingconducted. Activity may be assayed in a cell-free system, and generallywill comprise monitoring oxidation or prevention of oxidation ofproteins or lipids. Specifically, one will measure protection ofglutamine synthase from inactivation after addition of thiol-containingreducing agents (e.g., DTT) and metal (e.g., Fe⁺³). Activity also can bemeasured in a cell-free system by virtue of AOP2's ability to block O₂consumption when incubated with thiol-containing reducing agent andmetal ion (Netto et al., 1996).

A. In Vitro Assays

[0234] In one embodiment, the invention is to be applied for thescreening of compounds that bind to the AOP2 molecule or fragmentthereof. The polypeptide or fragment may be either free in solution,fixed to a support, expressed in or on the surface of a cell. Either thepolypeptide or the compound may be labeled, thereby permittingdetermining of binding.

[0235] In another embodiment, the assay may measure the inhibition ofbinding of AOP2 to a natural or artificial substrate or binding partner.Competitive binding assays can be performed in which one of the agents(AOP2, binding partner or compound) is labeled. Usually, the polypeptidewill be the labeled species. One may measure the amount of free labelversus bound label to determine binding or inhibition of binding.

[0236] Another technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with AOP2 and washed.Bound polypeptide is detected by various methods.

[0237] Purified AOP2 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to immobilize the polypeptideto a solid phase. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link the AOP2 activeregion to a solid phase.

[0238] Various cell lines containing wild-type or natural or engineeredmutations in AOP2 can be used to study various functional attributes ofAOP2 and how a candidate compound affects these attributes. Methods forengineering mutations are described elsewhere in this document, as arenaturally-occurring mutations in AOP2. In such assays, the compoundwould be formulated appropriately, given its biochemical nature, andcontacted with a target cell. Depending on the assay, culture may berequired. The cell may then be examined by virtue of a number ofdifferent physiologic assays. Alternatively, molecular analysis may beperformed in which the function of AOP2, or related pathways, may beexplored. This may involve assays such as those for protein expression,enzyme function, substrate utilization, phosphorylation states ofvarious molecules including AOP2, cAMP levels, mRNA expression(including differential display of whole cell or polyA RNA) and others.

[0239] One also may employ constructs where reporter genes are linked toAop2 coding sequences or regulatory sequences. In this way, the increasein expression may be measured by looking at a distinct function,associated with the reporter, that is easier to measure than, forexample, antioxidant activity. Examples of reporter genes include CAT,β-galactosidase, luciferase and green fluorescent protein.

B. In Vivo Assays

[0240] The present invention also encompasses the use of various animalmodels. Here, the high degree of homology seen between human and mouseAOP2 provides an excellent opportunity to examine the function of AOP2in a whole animal system where it is normally expressed. By developingor isolating mutant cells lines that fail to express normal AOP2, onecan generate animal models that will be highly predictive of diseases,including atherosclerosis, in humans and other mammals. Finally,transience or congenic animals (discussed below) that lack a wild-typeAOP2 may be utilized as models for atherosclerosis development andtreatment.

[0241] Treatment of animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that can be utilized for clinical ornon-clinical purposes, including but not limited to oral, nasal, buccal,rectal, vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

[0242] Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, increased activity level, reduced oxidation ofprotein or lipid, lowered HDL level, reduced oxidative products, reducedatherosclerosis, reduced oxidized LDL, reduced aging and reduced brainpathology.

C. Rational Drug Design

[0243] The goal of rational drug design is to produce structural analogsof biologically active polypeptides or compounds with which theyinteract (agonists, antagonists, inhibitors, binding partners, etc.). Bycreating such analogs, it is possible to fashion drugs which are moreactive or stable than the natural molecules, which have differentsusceptibility to alteration or which may affect the function of variousother molecules. By virtue of the availability of cloned Aop2 sequences,sufficient amounts of AOP2 can be produced to perform crystallographicstudies. In addition, knowledge of the polypeptide sequences permitscomputer employed predictions of structure function relationships. Inone approach, one would generate a three-dimensional structure for AOP2or a fragment thereof. This could be accomplished by x-raycrystallography, computer modeling or by a combination of bothapproaches. An alternative approach, “alanine scan,” involves the randomreplacement of residues throughout molecule with alanine, and theresulting affect on function determined.

[0244] It also is possible to isolate a AOP2-specific antibody, selectedby a functional assay, and then solve its crystal structure. Inprinciple, this approach yields a pharmacore upon which subsequent drugdesign can be based. It is possible to bypass protein crystallographyaltogether by generating anti-idiotypic antibodies to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of anti-idiotype would be expected to be an analog ofthe original antigen. The anti-idiotype could then be used to identifyand isolate peptides from banks of chemically- or biologically-producedpeptides. Selected peptides would then serve as the pharmacore.Anti-idiotypes may be generated using the methods described herein forproducing antibodies, using an antibody as the antigen.

[0245] Thus, one may design drugs which have improved AOP2 activity orwhich act as stimulators, inhibitors, agonists, antagonists of AOP2 ormolecules affected by AOP2 function. Another drug design approachrelates to the functional aspects of AOP2, namely, its putative role asan antioxidant. Using compounds that are antioxidants, that promoteantioxidant activity or that prevent oxidate directly, one may designnew compounds that function in conjunction with AOP2.

VII. Methods for Treating AOP2 Related Disease States

[0246] The present invention also involves, in another embodiment, thetreatment of atherosclerosis. This treatment may comprise provision ofAOP2 or provision of an expression construct containing an Aop2 gene.

A. Genetic Based Therapies

[0247] One of the therapeutic embodiments contemplated by the presentinventors is the intervention, at the molecular level, in the eventsinvolved in the development of atherosclerosis. Specifically, thepresent inventors intend to provide, to an appropriate target cell, anexpression construct capable of providing AOP2 to that cell. The lengthydiscussion of expression vectors and the genetic elements employedtherein is incorporated into this section by reference. Particularlypreferred expression vectors are viral vectors such as adenovirus,adeno-associated virus, herpesvirus, vaccinia virus and retrovirus. Alsopreferred is liposomally-encapsulated expression vector.

[0248] Those of skill in the art are well aware of how to apply genedelivery to in vivo and ex vivo situations. For viral vectors, onegenerally will prepare a viral vector stock. Depending on the kind ofvirus and the titer attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles tothe patient or animal. Similar figures may be extrapolated for liposomalor other non-viral formulations by comparing relative uptakeefficiencies. Formulation as a pharmaceutically acceptable compositionis discussed below.

B. Protein Therapy

[0249] Another therapy approach is the provision, to a subject, of AOP2polypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

C. Combined Therapy with Traditional Anti-Atherosclerotic Therapy

[0250] One goal of current research is to find ways to improve theefficacy of pharmaceuticals that are designed to reduce atherosclerosis.One way is by combining such traditional therapies with gene therapy. Inthe context of the present invention, it is contemplated that Aop2replacement therapy could be used similarly in conjunction withtraditional pharmaceutical intervention.

[0251] Generally, one will contact a “target” cell with an Aop2expression construct and at least one other agent. These compositionswould be provided in a combined amount effective to reduce theatherosclerotic burden of the animal. This process may involvecontacting the cells with the expression construct and the agent(s) orfactor(s) at the same time. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth agents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theexpression construct and the other includes the agent.

[0252] Alternatively, the gene therapy treatment may precede or followthe other agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

[0253] It also is conceivable that more than one administration ofeither an Aop2 expression construct or the other agent will be desired.Various combinations may be employed, where Aop2 is “A” and the otheragent is “B”, as exemplified below: A/B/A B/A/B B/B/A A/A/B B/A/A A/B/BB/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/AA/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

[0254] Other combinations are contemplated.

[0255] Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that reduces the atheroscleroticburden on a host or alters cholesterol or lipoprotein levels. The agentmay be prepared and used as a combined therapeutic composition, or kit,by combining it with a Aop2 expression construct, as described above.

[0256] The skilled artisan is directed to “Remington's PharmaceuticalSciences” 15th Edition, chapter 33, in particular pages 624-652. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

[0257] The inventors propose that the regional delivery of Aop2expression constructs to patients with AOP2-linked diseases will be avery efficient method for delivering a therapeutically effective gene tocounteract the pathological disease. Similarly, the therapy may bedirected to a particular, affected region of the subject's body.Alternatively, systemic delivery of expression construct and/or theagent may be appropriate in certain circumstances occurred.

D. Formulations and Routes for Administration to Patients

[0258] Where clinical applications are contemplated, it will benecessary to prepare pharmaceutical compositions—expression vectors,virus stocks, proteins, antibodies and drugs—in a form appropriate forthe intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

[0259] One will generally desire to employ appropriate salts and buffersto render delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

[0260] The active compositions of the present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra.

[0261] The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0262] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0263] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0264] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0265] For oral administration the polypeptides of the present inventionmay be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0266] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0267] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

VIII. Congenic, Transgenic and Knockout Animals

[0268] In one embodiment of the invention, congenic animals are producedthat have “normal” or mutant Aop2 genes. These animals will find use intesting of compounds in vivo that stimulate or inhibit AOP2 expressionor function. For example, compounds that improve the antioxidantactivity of AOP2 can be identified using these congenic animals, using avariety of different functional readouts, as described above. Methodsfor breeding congenic strains are well known to those of skill in theart.

[0269] In another embodiment, transgenic animals are produced whichcontain a functional or non-functional (e.g., mutant) transgene encodinga functional AOP2 polypeptide or variants thereof. Transgenic animalsexpressing Aop2 transgenes, recombinant cell lines derived from suchanimals and transgenic embryos may be useful in methods for screeningfor and identifying agents that induce or repress function of AOP2.Transgenic animals of the present invention also can be used as modelsfor studying indications such as atherosclerosis.

[0270] In one embodiment of the invention, an Aop2 transgene isintroduced into a non-human host to produce a transgenic animalexpressing a human or murine Aop2 gene. The transgenic animal isproduced by the integration of the transgene into the genome in a mannerthat permits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. 1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

[0271] It may be desirable to replace the endogenous Aop2 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, an Aop2 gene flanked by genomicsequences is transferred by microinjection into a fertilized egg. Themicroinjected eggs are implanted into a host female, and the progeny arescreened for the expression of the transgene. Transgenic animals may beproduced from the fertilized eggs from a number of animals including,but not limited to reptiles, amphibians, birds, mammals, and fish.Within a particularly preferred embodiment, transgenic mice aregenerated which overexpress AOP2 or express a mutant form of thepolypeptide. Alternatively, the absence of a Aop2 in “knock-out” micepermits the study of the effects that loss of AOP2 protein has on a cellin vivo. Knock-out mice also provide a model for the development ofAOP2-related diseases.

[0272] As noted above, transgenic animals and cell lines derived fromsuch animals may find use in certain testing experiments. In thisregard, transgenic animals and cell lines capable of expressingwild-type or mutant AOP2 may be exposed to test substances. These testsubstances can be screened for the ability to enhance wild-type AOP2expression and or function or impair the expression or function ofmutant AOP2.

IX. EXAMPLES

[0273] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those ofskilled the art that the techniques disclosed in the examples whichfollow represent techniques discovered by the inventor to function wellin the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 1—Materials and Methods

[0274] Mice. C57BL/6J, DBA/2J, and 8 BXD recombinant inbred strains (BXD1, BXD 2, BXD 5, BXD 6, BXD 8, BXD 9, BXD 11, BXD 25) were obtained fromThe Jackson Laboratory (Bar Harbor, Maine).

[0275] Two dimensional gel electrophoresis: Proteins were extracted fromkidney and liver by a modification of the method of Wilson et al.(1977). 200 mg of tissue were homogenized on ice in 68 μl of lysisbuffer containing 10% mercaptoethanol, 1% SDS, 8 M urea. Five hundred μlof boiling sample buffer 1 (0.3% SDS, 200 mM DTT, 28 mM Tris HCl, 22 mMTris-base) was added followed by incubation at 100° C. for 5 min.Samples were chilled on ice for 5 min. mixed with 50 μl of sample buffer2 (24 mM Tris-base, 476 mM Tris-HCl, 50 mM MgCl2, 1 mg/ml DNAase I, 25mg/ml RNAse A), incubated on ice for 8 min. and proteins wereprecipitated with 80% acetone at 0° C. for 20 min. The suspension wasmicrocentrifuiged (10,000 g, 5 min.) and the precipitate was resuspendedin 500 μl of loading buffer (7.92 M urea, 0.06% SDS, 1.76% Ampholytessolution pH 3-7, 120 mM DTT, 3.2% Triton X-100, 22.4 mM Tris HCl and17.6 mM Tris-base). Fifteen μl of each sample (approximately 10 μg ofprotein) was electrophoresed. Analytical and preparative two-dimensionalelectrophoresis were performed using apparatus, protocols and 2-D gradereagents purchased from the Millipore Corp. Isoelectric point andmolecular weight standards (BioRad) were run simultaneously withanalyzed protein samples.

[0276] Protein blotting and protein microsequencing. Proteins weretransferred from preparative two-dimensional electrophoresis gels usingthe MilliBlot Graphite Semi-Dry Electroblotter System and Immobilon-P(PVDF) Transfer Membrane (Millipore) in 0.5×Tris-Glycine Towbin bufferaccording to protocol provided by manufacturer. After transfer themembrane was stained with 0.25% Coomassie brilliant blue, and theprotein spot was identified and excised from dry membrane. N-terminalamino acid sequence was obtained using an Applied Biosystems Model 477Aprotein sequencer, modified as described by Tempst and Riviere²⁴.Homology searches were performed against the Genpet and dbEST databases.Sequence alignment and consensus analysis of the EST sequences was doneusing Sequencher 3.0 (Gene Codes). Genbank accession numbers for ESTsused for sequence alignment include: W70913, W71818, W14400, W51064,W59407, W83606, W83464, AA002970. Terminal 3′ UTR sequence was obtainedusing Sequenase (USB) according to the manufacturer's instructions.

[0277] SSCP mapping. Single-stranded conformational polymorphism (SSCP)analysis of the 3′ UTR of the presumptive Ltw4 gene was employed forgenetic mapping (Guertin et al., 1994; Hahn & Subbiah, 1994). Apolymorphism between the C57BL/6J and M. spretus strains was identifiedusing PCR amplification of the primers: 5′-GCGAGCGATCTACAGGACC-3′ (SEQID NO: 9) and 5′-TGATGGTAGTTCCCACCCTC-3′ (SEQ ID NO: 10)

[0278] Mapping was done using the panel of 94 DNAs from the BSSinterspecific backcross (lakoubova et al., 1997) as follows: 30 ng ofDNA was amplified in 96 wells microtiter plates with ³²P end-labeledforward and reverse primers (0.3 μM of each primer) in total volume of12 μl of PCR buffer containing 10 mM Tris pH 8.0, 50 mM KCL, 2 mM MgCl₂and 0.001% gelatin. After denaturation at 94° C. for 5 min. 0.2 mM dNTPsand 0.5 units of Taq DNA polymerase were added. Twenty-five cycles ofamplification (55° C. annealing for 2 min. 72° C. extension for 3 min.94° C. denaturation for 1 min.) were performed with a final extensionstep of 7 min. PCR products were mixed 1:4 with USB stop solution,denatured at 95° C., transferred on ice and 2 μl of each sampleelectrophoresed through a 5% non-denaturing polyacrylamide gel in0.5×TBE buffer at 4° C., 40 W constant power for 2-3 hr. The straindistribution pattern was analyzed using the Map Manager computerprogram.

Example 2—Results

[0279] LTW4 protein was identified by surveying approximately onethousand silver-stained protein spots obtained after two-dimensionalelectrophoresis of liver and kidney homogenates for a polymorphicprotein similar to that originally described by Elliott, 1979. Apolypeptide of 26 kD was identified whose C57BL/6J allele had a pI of5.9 and whose DBA/2J allele had a pI of 5.6 (FIG. 1). To ensure that thepolymorphic protein identified corresponded to LTW4, 8 BXD RI strainswere analyzed using two-dimensional gel electrophoresis, and determinedthat the strain distribution pattern of this protein in these strainswas identical to that described for Ltw4.

[0280] Preparative 2-D electrophoresis was performed using liverproteins extracted from DBA/2J strain, since this variant of LTW4 wasmore readily resolved from other proteins than the C57BL/6J-relatedisoform. After electrophoresis in the second dimension, proteins weretransferred to a membrane and the LTW4 protein spot identified andisolated. Microsequencing of the protein spot yielded twenty-fourN-terminal amino acids that had 92% identity with residues 2-26 of apreviously identified human hypothetical protein (HUMORF06, Genbankaccession number: D14662) which appears to be a member of a large familyof thiol-specific antioxidant (TSA) proteins (Elliott et al., 1980). Thenucleotide sequence of HUMORF06 was used to identify homologous murinepartial cDNA sequences using BLASTN searches against the dbEST database.A consensus sequence from these ESTs was obtained using Sequencher 3.0sequence alignment software (FIG. 2). The translated product of thisconcensus sequence has 89% identity and 93% similarity with HUMORF06. Asdiscussed below, another TSA family member, the murine MER5 gene,recently renamed antioxidant protein 1 (Aop1), has regions of conservedhomology with the gene characterized here; therefore, this gene has beendesignated antioxidant protein 2 (Aop2).

[0281] SSCP analysis was used as previously described to assess whetherthe gene encoding the polymorphic protein isolated maps to the sameposition as Ltw4 (Guertin et al., 1994; Hahn & Subbiah, 1994). An SSCPbetween C57BL/6J and M. spretus was identified using primers derivedfrom 3′ UTR of Aop2 and these primers were then tested in the BSSinterspecific backeross (FIG. 3A & B) (Iakoubova et al., 1997). Aop2 waslocalized to chromosome 1 with a LOD likelihood of 27.4. No recombinantswere observed between Aop2 and Pmx1 in 91 backcross progeny, and theposition of these loci with respect to flanking microsatellite markersis D1Mit14−3.2±1.8 cM-(Aop2, Pmx1) −2.2±1.5 cM-D1Mit110. The mapposition of Ltw4 in the BXD RI series is D1Mit111 −23±11cM−(Ltw4,At3)−3.1±3.2 cM−D1Mit16. Since D1Mit16 is 1.1 cM proximal toD1Mit110, the position of Aop2 and Ltw4 with respect to these distal Mitmicrosatellite markers is within the same 95% confidence interval. Inaddition, At3, which is nonrecombinant with Ltw4 in the BXD series, mapsbetween D1Mit 14 and D1 Mit110 (Marcel et al., 1989). Finally, theregion of chromosome 1 in which Ltw4 has been mapped demonstratesconservation of synteny with human chromosome 1q23-25. WI-7237, an STSderived from HUMORF06 (the human homologue of Aop2), has been mapped inthis region on radiation hybrid and YAC physical maps (Ohkawa et al.,1979). These mapping studies confirm that Aop2 corresponds to Ltw4.

Example 3—Discussion

[0282] LTW4 protein shares amino acid similarity with members of a classof thiol-specific antioxidant (TSA) proteins that have been identifiedin a wide variety of organisms (Elliott et al., 1980). The bestcharacterized of these proteins include yeast TSA, which has been shownto protect cellular components against oxidative damage from a systemcapable of generating reactive sulfur species, but not from a systemthat generates only reactive oxygen (Paigen et al., 1987a). Thisactivity is also found for AhpC, the catalytic component of the alkylhydroperoxide reductase from S. typhimurium (also called C22) (Elliottet al., 1980). The mammalian members of this protein family includebrain TSA (Elliott et al., 1980), ER5 (Paigen et al., 1987b),osteoblast-specific factor 3 (OSF3) (Paigen et al., 1987c), naturalkiller enhancer factors A and B (NKEFA and NKEFB) (Paigen et al., 1990),proliferation-associated gene (PAG) (Parthasarathy et al., 1990), andmacrophage 23 kD stress protein (MSP23) (Senault et al., 1990). (OSF3,NKEFA, PAG and MSP23 are highly homologous and may be variants of thesame protein.) Amino acid comparison of AOP2, MER5, MSP23, and TSA usingthe Blocks program (http://www.blocks.fhcrc.org) reveals three domainsof conservation (FIG. 4). It is apparent that AOP2 is the least similarof these proteins; of particular interest is that AOP2 does not sharethe second cysteine that is conserved in the other three murine proteinsas well as in most members of the TSA gene family (Elliott et al.,1980). While the specific functions of these genes have not yet beenelucidated, it is of note that MSP23 was initially isolated as a proteinthat was induced in response to oxidative stress. In addition, it hasbeen shown that the gene encoding MER5 can complement a deficiency ofalkyl hydroperoxide reductase activity in E. coli that is due to amutation in AhpC (Yamamoto et al., 1989). Based upon this result, theMer5 gene has been renamed antioxidant protein 1 (Aop1).

[0283] The protein encoded by Aop2 was originally found to be anabundant product in homogenates of liver, kidney, and brain (Chae etal., 1994; Elliott, 1979). Aop2 cDNA appears to be widely expressed: theTIGR database (http://www.tigr.org/tdb/tdb.html) identifies a tentativehuman concensus (THC122873) that corresponds to HUMORF06 (the humanhomologue of Aop2), this THC is comprised of 116 ESTs that have beenidentified in cDNA libraries derived from 24 different tissues.

Example 4—Materials and Methods

[0284] SSCP mapping. Single-stranded conformational polymorphism (SSCP)analysis of the 3′ untranslated region of the Aop2 gene was employed forgenetic mapping in the congenic Athl strains (Beier et al., 1992; Beier,1993). A polymorphism between the C57BL/6J and M. spretus strains wasidentified using PCR amplification of the primers:5′-GCGAGCGATCTACAGGACC-3′ (SEQ ID NO: 9) and5′-TGATGGTAGTTCCCACCCTC-3′ (SEQ ID NO: 1O)

[0285] Mapping was performed using the BSS Jackson Laboratory panel ofbackcross mice derived from backcross of (C57BL/6 X Spretus) F1 micebackcrossed to Spretus as follows: 30 ng of DNA was amplified in 96-wellmicrotiter plates with 0.3 mM ³²P end-labeled forward and reserveprimers in a total volume of 12 ml of PCR buffer containing 10 mM TrispH 8.0, 50 mM KCL, 2mM MgCl₂ and 0.001% gelatin. After denaturation at94° C. for 5 min., 0.2 mM dNTPs and 0.5 units of Taq DNA polymerase wereadded. Twenty-five cycles of amplification (55° C. annealing for 2 min,72° C. extension for 3 min, 94° C. denaturation for 1 min) wereperformed with a final extension step of 7 min. PCR products were mixed1:4 with USB stop solution, denatured at 95° C., transferred to ice and2 ml of each sample electrophoresed through a 5% non-denaturingpolyacrylamide gel in 0.5×TBE buffer at 4° C., 40 W constant power for2-3 hr. Using this approach, mice heterozygous for C57BL/6J and M.spretus alleles could be distinguished from mice homozygous for theC57BL/6J allele. Homozygosity for the C57BL/6J allele of Aop2 andsusceptibility to atherosclerosis were concordant in 100% of these mice.This corresponds to a genetic distance of 0-x cM.

[0286] Sequence analysis: Clones containing full-length open readingframes were obtained using RT-PCR amplification from cDNA prepared fromkidney and liver of C57BL/6J, DBA/2J, and C3H/FeJ mice according tostandard techniques. Primers used for this amplification were AOP2 orfforward: 5′-AGCGTCACCACTGCCGCCATG-3′ (SEQ ID NO:11) and AOP2 orfreverse: 5′-GTACTGGATGTGCAGATGCAGCC-3′ (SEQ ID NO:12). Multipleindependent PCR reactions were done for each strain and the amplifiedfragment cloned into pCR2.1 (Invitrogen) and sequenced using Sequenase(USB) according to the manufacturer's instructions.

Example 5—Results

[0287] Construction of the congenic strain. In order to construct acongenic strain with susceptible C57BL/6 (B6) as the host strain, andthe resistant allele of Athl from another strain with more polymorphicdifferences from B6 than the existing congenic B6.C-H25^(c)/By, a regionof the Spretus genome was introduced into B6 as these two strains arefrom different, although closely related, species. The Athl region fromSpretus was backcrossed into B6 using the markers D1Mit14 and D1Mit136for 5 generations and then intercrossing to obtain the F1 generation.The attempt to breed the NSF1 generation to homozygosity failed;homozygous F2 mice were obtained but did not breed to N5F2. The F2 malesapparently have a deficiency of sperm, and these sperm do not fertilizeeggs even by in vitro fertilization. The heterozygote congenic is beingpreserved as frozen embryos.

[0288] High resolution mapping of Athl. To map Athl with highresolution, a backcross was carried out between C57BL/6 and theheterozygous congenic strain B6.Spret-Athl^(r/s). This cross included1176 progeny, and 89 crossovers were obtained between the markersD1Mit14 and D1Mit356, which flank the Athl region. Mice carryingcrossovers were mated to B6 and its female progeny collected and fed thehigh fat diet for 18 weeks. Lesion size was determined for each mouseand averaged over all progeny from each crossover parent to determinewhether the parent carried the resistant or susceptible allele of Athl.Polymorphic genetic loci were typed in the crossover animals; the highresolution map is depicted in FIG. 5. Each crossover represents agenetic distance of 1763/100 or 0.057 cM.

[0289] No recombinant mice were found between Athl and the SSLP lociD1Mit105, D1Mit266, and D1Mit424. Since Aop2 mapped to this region ofmurine chromosome 1, primer pairs were utilized that recognize apolymorphic difference in the 3′ -untranslated region of Aop2. Thesewere mapped in a panel of mice that carried unique crossovers in progenyof the high resolution backcross. No recombinants were found betweenAthl and Aop2, suggesting that Aop2 is Athl.

[0290] Sequencing Athl/Aop2 cDNA. Primers flanking the presumptivecoding region of Athl/Aop2 were utilized to amplify and clone thisregion from kidney and liver cDNA prepared from the C57BL/6J, DBA/2J,and C3H/FeJ inbred mouse strains; the sequence of these clones appearsin FIG. 2. The C57BL/6J and DBA/2J alleles of this gene code forproteins with a single amino acid difference: amino acid 122 correspondsto aspartic acid in DBA/2J and to alanine in C57BL/6J. The translatedproduct of the full length coding sequence has 89% identity and 93%similarity with a human sequence HUMORF06. The protein encoded by Aop2was originally found to be an abundant product in homogenates of liver,kidney, and brain (Elliott, 1979; Racine and Langley, 1980; Goldman andPikus, 1986).

Example 6—Discussion

[0291] Aop2 was initially characterized as a candidate for the Jckm2modifier locus. The possible role of an antioxidant protein in theprogression of polycystic kidney disease is not obvious. It is of note,however, that Aop2 maps in the region identified for atherosclerosis 1(Athl), a locus that has been suggested to play a role in determiningthe difference in the relative susceptibility of the C57BL/6J andC3H/HeJ mouse strains to the development of atherosclerotic plaques whenfed a high fat diet. The role of lipid oxidation in the pathogenesis ofatherosclerosis is well known, and recent studies have implicated Athlin either the accumulation of lipid peroxides in tissues, or thecellular response to such lipid peroxides. Since the Aop2 proteinproduct is polymorphic between C57BL/6J and C3H/HeJ, and since thebiochemical functions of other members of this gene family include thereduction of peroxides (lakoubova et al., 1997), Aop2 should wasconsidered the candidate for Athl. Aop2 cDNA appears to be widelyexpressed. The TIGR database (http://www.tigr.org/tdb/tdb.html)identifies a tentative human concensus (THC122873) that corresponds toHUMORF06. the human homolog of Aop2. This THC is comprised of 116 ESTsthat have been identified in cDNA libraries derived from 24 differenttissues.

Example 7

[0292] As discussed above, Athl is associated with reduced plasma HDLlevels and increased atherosclerotic lesion formation in susceptiblemice when fed a high fat diet, as compared to Athl resistant mice whichexhibit no change in plasma HDL levels and the absence ofatherosclerotic lesions. The mapping of this locus to chromosome 1 cM83.6 in the mouse genome coincided with the cloning and mapping of Aop2.Based on amino acid homology, Aop2 appears to encode a new member alarge family of thiol-specific antioxidants (TSA), which are highlyconserved (Chae et al., Proc. Natl. Acad. Sci. USA 91: 7017-7021(1994)). These proteins possess activity which protects cells fromoxidative damage resulting from metal-catalized oxidation systems, andare thus thought to be an important part of the complex cellular defenseagainst oxidative stress. Aop2 cDNA was isolated from liver and kidney,but was also found to be expressed in a wide variety of tissues(lakoubova et al., Genomics 42: 474-478 (1997)). This gene was found toencode the previously characterized Ltw-4 protein, which was shown to bepolymorphic between the C57BL/6J and DBA/2J strains of mice.

[0293] The cDNA corresponding to Aop2 has been independently isolated bytwo other laboratories, one of which has called it a glutathioneperoxidase due to its high identity a bovine ciliary glutathioneperoxidase (Frank et al., Oncogene 14: 915-921 (1997); Munz et al., J.Biochem. 326: 579-585 (1997)), and the other has called it aphospholipase for the presence of a “GXSXG” motif, which is reminiscentof serine hydrolase activity (Kim et al., J. Biol. Chem. 272: 2542-2550(1997)). However, Aop2 shows no significant homology with any otherknown glutathione peroxidases, nor does it resemble any phospholipasesthat have been identified. In contrast, Aop2 demonstrates significanthomology with over 50 known thiol-specific antioxidants. Furthermore,TSA activity has in fact been demonstrated for the human homolog ofAop2. Therefore, Aop2 appears to be a member of the TSA gene family.Since LDL oxidation has been implicated in foam cell formation andatherosclerotic lesion development in the arterial wall, Aop2 wasinvestigated as a candidate gene for Athl.

[0294] The Athl locus was previously mapped to a 3 cM region onchromosome 1 using recombinant inbred strains. Since there wereinsufficient polymorphic markers between B6 and C3H, two independentcongenics were made, which carried the Athl allele from theatherosclerosis-resistant wild-derived strains, Spretus and PERA, on aC57BL/6J background. Using a congenic on a B6 background avoids theproblems of all the other genes in these wild-derived strains thataffect HDL and atherosclerosis, but adds the benefit of many polymorphicmarkers in the region of Athl. Backcross progeny were collected from theN5 incipient congenic derived from Spretus; theoretically the congenicat the fifth generation has only about 3% of Spretus gene except for theselected region on Chromosome 1. Therefore, the chance that otherSpretus genes besides Athl would affect HDL-C and lesions in a crosswith B6 is greatly reduced. 1763 backcross progeny were collected fromthis cross and tested for progeny with recombination events betweenD1Mit14 and D1Mit356, a genetic distance of 10 cM based on the MIT map.A total of 162 recombinant mice were accumulated and analyzed in a twostep process. Step 1: Female recombinants were tested for lesions,accepting the fact that one can not rely on the phenotype from eachindividual mouse due to the high variability in this quantitative trait.This narrowed the region to D1Mit14- -D1Mitl 10, a 3-4 cM region. Step2: Male mice with a recombination event in this 3-4 cM region were bred,and female progeny tested for atherogenic phenotype. This progenytesting avoids the problem of high variability by testing groups ofprogeny mice to determine the phenotype of a single recombinant mouse.This two step procedure narrowed the region considerably. Eachrecombinant equals a distance of 1/1763 or 0.05 cM. If 1 cM equals 1.75megabases, the physical distance between each crossover can be estimatedto be about 87.5 kb. Genes are estimated to occur on the average every40 kb (although there are gene rich and gene poor regions). A finestructure map of this region reveals that Athl shows no recombinationwith D1Mit105, D1Mit266 and D1Mit424, and lies in a region betweenD1Mit159 and D1Mit398 that is 0.15 cM in length. This excludes, as acandidate, apolipoprotein A2, whose map position is below this region.It also excludes Acact (acylcoenzyme A:cholesterol acytransferase), thegene coding for the enzyme ACAT, which metabolizes cholesterol to astorage molecule, cholesterol ester, which maps above this region.

[0295] Since it was previously shown that Aop2 is polymorphic betweenC57BL/6J and Spretus, the 37 mice that were recombinant in the Athlregion were directly assayed for which Aop2 allele they carried. Theseresults showed no recombination with Athl. Because an antioxidantprotein is an attractive candidate gene for atherosclerosis, the nextsteps are to determine if Aop2 is in fact Athl.

[0296] In order to determine if resistant and susceptible phenotypescorrelate with different Aop2 alleles, Aop2 cDNA was amplified andsequenced from the susceptible C57BL/6J strain, fouratherosclerosis-resistant inbred strains (C3H/HeJ, BALB/cJ, DBA and129/SvJ), and the two atherosclerosis-resistant wild strains, Spretusand PERA, used to independently derive the Athl congenics. Thenucleotide sequences corresponding to the coding region of Aop2, and thecorresponding amino acid sequences were determined in each strain. Allfour resistant inbred strains possessed the single amino acid difference(at amino acid#124) from the susceptible B6 strain. This amino acid isalanine in the susceptible C57BL/6J strain, and aspartic acid in each ofthe four resistant inbred strains. Although the functional significanceof this amino acid difference is not known, prior analysis of thisdifference has revealed that it can account for the difference in the 2Dgel migration pattern of Aop2 protein previously reported between the B6and DBA strains. In contrast, the two atherosclerosis resistant wildstrains that were analyzed, PERA and Spretus, do not possess this aminoacid difference and appear to carry the same Aop2 allele as thesusceptible C57BL/6J. Therefore, the mere presence of this amino aciddifference could not explain resistance in all strains. For this reason,the characterization of Aop2 mRNA expression was undertaken to determineif these strains exhibited differences in Aop2 expression.

[0297] Aop2 mRNA levels were compared from liver tissue of thesusceptible B6 strain, the resistant BALB/cJ strain, and the tworesistant Athl congenic strains, B6. PERA and B6. Spretus, on both chowor high fat diet for 4 or 10 weeks. Total RNA was isolated fromindividual livers, and RNA from two individual mice, and a pool of 10mice were analyzed from each strain on both chow or high fat diet. Aop2expression in the liver was similar on chow and high fat diet in thoseresistant strains possessing the Aop2 allele carrying aspartic acid atamino acid position 124. In contrast, all strains carrying the Aop2allele encoding alanine at amino acid position 124 showed a significantinduction of Aop2 on high fat diet. Induction occurred in all threestrains by 4 weeks on diet. However, the level of Aop2 expression onboth chow and high fat diet was significantly greater in the resistantstrains, as compared to the susceptible C57BL/6J.

[0298] To further support the conclusion that Aop2 is Athl, acorrelation between Athl phenotype and Aop2 expression in criticalcrossover animals put on high fat diet was investigated. The presence orabsence of atherosclerotic lesions in these individuals was comparedwith their Aop2 expression in the liver. The two resistant B6. Spretuscongenic animals (N6) showed high levels of Aop2 expression after 14weeks on high fat diet. These animals did not get lesions. In addition,4 crossover animals, which lacked lesions, and four which developedatherosclerotic lesions by 14 weeks on high fat diet, were directlycompared. Those animals which were resistant to lesions expressed higherlevels of Aop2 after 14 weeks on high fat diet than did those whichdeveloped lesions. Therefore, there is a correlation between developmentof atherosclerotic lesions and expression level of Aop2.

Example 8—Materials & Methods (a) Isolation and Subcloning of Aop2Genomic Clones.

[0299] To isolate a genomic clone for the mouse Aop2 gene, a 0.7 kb cDNAfragment spanning the entire coding region of the murine Aop2 cDNA wasamplified from the DBA mouse strain by PCR using Aop2orf forward(5′-AGCGTCACCACTGCCGCCATG-3′) and Aop2orf reverse(5′-GTACTGGATGTGCAGATGCAGCC-3′) primers. This fragment was isolated,32P-radiolabeled by random priming (Amersham, RediPrime Kit), and usedas a probe to screen a 129/SvJ IFIXRII genomic library (Stratagene, LaJolla, Calif., USA). Approximately 1.8×10⁶ plaques were screened.Individual positive clones were selected after a second round ofscreening, and phage DNA was isolated from each clone using standardtechniques. Southern blotting was performed on EcoRI digested genomicclones using the ³²P-radiolabeled Aop2 cDNA probe to distinguish uniquesequences. Preliminary sequencing was performed on lambda clones usingthe dideoxy chain termination method with both the M13 forward andreverse primers, and specific primers designed according to thepublished Aop2 cDNA sequence (lakoubova et al, Genomics 42: 474-478(1997)). Positive clones were digested with NotI, subcloned into thepBluescript SK—vector (Stratagene, La Jolla, Calif., USA) and weresequenced using the dideoxy chain termination method (USB).

(b) Determination of Intron/Exon Junctions, Intron Size, & PromoterSequence

[0300] The intron/exon junctions of Aop2 were determined by sequencingof subcloned genomic fragments using exon-specific primers flanking eachintron. These included: Exon1F (5′GAGGATTGCTTCTCGGGG-3′), Exon2R(5′-CCGTGGGTGGGAAAAGAG-3′), Exon2F (5′-CATTCTCTTTTCCCAC-3′), Exon3R(5′-TTTCCGTGGGTGTTTCAC-3′), Exon4F (5′-CCTCTACCCTGCCAC CAC -3′), Exon5R(5′-ACCATCACGCTCTCTCCC-3′). All introns except intron#4, which wassequenced completely, were sized by estimation of PCR product sizesusing the following combinations of primers: Exon1F & Exon2R; Exon2F &Exon3R; Exon 4F & Exon5R. PCR reactions were performed using standardconditions with 30 cycles of the following parameters: 94°, 1 min; 55°,1 min; 72°, 2 min. PCR products were separated on a 0.8% agarose gel andsizes were estimated based on comparison with lambda/Hind III andlambda/BstI molecular weight standards (Promega). The 5′ non-codingsequence was obtained using an exon1 reverse primer(5′-GTCCCCGAGAAGCAAACC-3′), the Exon2R primer, above, and an upstreamforward primer (5′-CCCACGTCACAAGTCTGG-3′) designed to the sequencedupstream region. The reported putative promoter sequence was confirmedby at least three separate sequencing reactions.

Results

[0301] A genomic library made from the 129/SvJ mouse strain was screenedusing a probe made from the coding region of the mouse Aop2 cDNA. Twentyone individual positive clones were identified, and Southern blottingwas used to distinguish unique sequences. The results from this analysissuggested at least three distinct genes; these included Aop2 and twohighly related intronless genes.

[0302] One group of phage clones contained overlapping sequences of thetrue Aop2 gene, as demonstrated by over 99% nucleotide identity to theAop2 cDNA isolated from the C57BL/6J and DBA/2J strains of mice. Theoriginal publication of the Aop2 cDNA reported two nucleotides in thecoding region that were divergent between the DBA/2J and C57BL/6Jstrains; one of which is at cDNA position#80, which does not change thecoded amino acid, and the second is at nucleotide#439, which is adeninein DBA/2J and cytosine in C57BL/6J. The corresponding amino acids atthis position are aspartic acid in DBA/2J and alanine in C57BL/6J. Likethat of DBA/2J the Aop2 gene from the 129/SvJ strain encodes asparticacid at nucleotide#439. The Aop2 allele from 129/SvJ differs from DBA/2Jonly at the variant nucleotide#80 which is guanine in 129/SvJ, encodingthe same amino acid as all of the other strains analyzed.

[0303] Further sequence analysis of these clones revealed that the geneconsists of five exons and four introns, spanning approximately 10.7 kb.The genomic structure of the mouse Aop2 gene is shown in FIG. 6. Thelocation of exons within the Aop2 gene are indicated in panel A. Asshown, all four introns are contained within the coding region of thegene. The precise size and location of exons and introns are shown inpanel B, as well as the confirmed sequence of the intron/exonboundaries. The nucleotide numbers correspond to the presumed fulllength Aop2 cDNA as previously reported (Munz et al., J. Biochem. 326:579-585 (1997); Frank et al., Oncogene 14: 915-921 (1997)). While theintron sizes range from 471 bp to approximately 3.5 kb in length, thefirst four exons are relatively close in size, ranging between 147 and163 bp. The 3′ end of the coding region and the 667 bp 3′ UTR are foundwithin the last exon. The 3′ UTR of Aop2 from the 129/SvJ strain wasalso compared with a partial 3′ UTR we have isolated from the C57BL/6strain, and the previously reported full length 3′UTR from BALB/cJ [Munzet al., J. Biochem. 326: 579-585 (1997), #2672]. We found a singlenucleotide difference at nucleotide #1037, which is thymine in C57BL/6Jand cytosine in 129/SvJ and BALB/cJ. These are the only strains forwhich the 3′ UTR of Aop2 is known.

[0304] In order to begin to understand how Aop2 may be regulated in vivothe region upstream of the 5′ most cDNA sequence, as previously reported(Munz et al., J. Biochem. 326: 579-585 (1997); Frank et al., Oncogene14: 915-921 (1997)), was isolated and sequenced. Approximately 500nucleotides of this upstream region are shown in FIG. 7. Analysis ofthis sequence identified no consensus TATA-box. However, severalpotential SP 1 binding sites are located within 60 nucleotides upstreamof the presumed +1 transcription start site, suggesting that basaltranscription of Aop2, like many other ubiquitously expressed genes, maybe regulated by these transcription factors. There is also a sequencewhich resembles a transcriptiation initiation sequence commonly found inTATA-less promoters (CTCANTCT). The actual sequence differs from thisconsensus by a single nucleotide insertion. However, this element islocated 20 nucleotides downstream of the presumed transcription startsite, as previously reported.

[0305] Several additional consensus recognition sequences for knowntranscription factors are found in the putative proximal promoter. Asshown in FIG. 7, these include potential binding sites for USF (upstreamstimulatory factor) and SREBP (sterol response element binding protein),two DNA binding proteins that have been demonstrated to be important inthe regulation of a number of genes involved in lipid metabolism. Theputative USF recognition sequence matches 100% to the known bindingsite, while the putative SREBP binding site contains 11 out of 12nucleotides of the consensus sequence. There is also a consensusrecognition sequence for ADR1 (alcohol dehydrogenase regulated gene 1),which is identical to the known binding site. In addition, this regioncontains several potential binding sites for Heat Shock Factor (HSF),all of which match perfectly to the consensus recognition sequence.

[0306] In addition to the Aop2 gene, two highly related genes were alsoidentified from the original screen. These genes have been namedAop2-rs1 and Aop2-rs2 (Aop2-related sequence 1 and 2). FIG. 8A displaysa comparison of the nucleotide sequences of all three genes,corresponding to the coding region of Aop2. As shown, Aop2-rs1 shares93% nucleotide identity with Aop2 in the coding region. Each nucleotidedifference in Aop2-rs1 is the result of a base substitution, resultingin several amino acid changes but an intact open reading frame. Incontrast, Aop2-rs2 appears much more divergent, sharing only 80%nucleotide identity with Aop2, including 67 nucleotide substitutions, 24nucleotide deletions and 39 nucleotide insertions. The first nucleotidedeletion interrupts codon# 114 and disrupts the reading frame, resultingin a truncated protein, which is 119 amino acids in length. An alignmentof the predicted protein products with the known Aop2 protein sequenceis shown in FIG. 8B. While the presumed Aop2-rs1 protein shows 86%identity to Aop2, Aop2-rs2 shares only 42% amino acid identity.

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1 12 1653 base pairs nucleic acid single linear 1 CGGTTGCTTG CTGTCCCAGCGGCGCCCCCT CATCACCGTC GCCATGCCCG GAGGTCTGCT 60 TCTCGGGGAC GTGGCTCCCAACTTTGAGGC CAATACCACC GTCGGCCGCA TCCGTTTCC 120 CGACTTTCTG GGAGACTCATGGGGCATTCT CTTCTCCCAC CCTCGGGACT TTACCCCAG 180 GTGCACCACA GAGCTTGGCAGAGCTGCAAA GCTGGCACCA GAATTTGCCA AGAGGAATG 240 TAAGTTGATT GCCCTTTCAATAGACAGTGT TGAGGACCAT CTTGCCTGGA GCAAGGATA 300 CAATGCTTAC AATTGTGAAGAGCCCACAGA AAAGTTACCT TTTCCCATCA TCGATGATA 360 GAATCGGGAG CTTGCCATCCTGTTGGGCAT GCTGGATCCA GCAGAGAAGG ATGAAAAGG 420 CATGCCTGTG ACAGCTCGTGTGGTGTTTGT TTTTGGTCCT GATAAGAAGC TGAAGCTGT 480 TATCCTCTAC CCAGCTACCACTGGCAGGAA CTTTGATGAG ATTCTCAGGG TAGTCATCT 540 TCTCCAGCTG ACAGCAGAAAAAAGGGTTGC CACCCCAGTT GATTGGAAGG ATGGGGATA 600 TGTGATGGTC CTTCCAACCATCCCTGAAGA AGAAGCCAAA AAACTTTTCC CGAAAGGAG 660 CTTCACCAAA GAGCTCCCATCTGGCAAGAA ATACCTCCGC TACACACCCC AGCCTTAAG 720 CTCTTGGAGA AGTTGGTGCTGTGAGCCAGA GGATGTCAGC TGCCAATTGT GTTTTCCTG 780 AGCAATTCCA TAAACACATCCTGGTGTCAT CACAGCCAAG GTTTTTAGGT TGCTATACC 840 ATGGCTTATT AAATGAAAATGGCACTAAAA GTTTCTTGAG ATTCTTTATA CTCTCTGCC 900 TCAGCAATCA ATTCCATTCATACATCAGCA CTCTGCTGGT TCTGTTTGAA ATATGTTCT 960 TATTTAAAAC TCAAATCTTGTTGGATCTCT GCAGGGCTTG TGACCAATGA AGTCATAT 1020 GTTGATGGTT GACAAAGCTTGCTTCACTCC ATCAGAGAAT GACTATCAAT TTTTTTTT 1080 CTGTCCTATC ACGTCCTCTCCTGTCACCCA TTTTGAAGAG TGGCAGAACT TGAAGTTC 1140 CTTCCTCTGT AAATATCCAAGTATAAAGCC CAGGAACTTC TAGAATAACC CAGATGCG 1200 TTAATTTTTT TTAATATGTTTTGATCACAG AACTTCTAGA ATAACCCAGA TGCTCTTT 1260 TATTCTTTTA ATACATCTTGATCACAGCTG GGGGAAAAAA AGCTTTTTAA TTCTGTAC 1320 TCCTAGTAGA TAAGTGAAGAGCAGGGAAAG AGACCTTTAA ATATTTTGCT ATAAAAAA 1380 TTGTGATAAG TTTCTATCAAAATGGGGAGA TTGCAGAAAA GGCTTCCCTT GGCTCCCA 1440 GAGGTGTAGC AGGTGTGAGCAATATTAGTG CCATGTGCCT TTCACACAGG GTTTGCAT 1500 ATCAGTCTGT TTTCCGATGATGTGTACATG AAAGAGTACA CCATGTGAAG AGAAGAGA 1560 ATGATTGAAA ATGTTTTAGTATAGAACTCT TCTTGCAGTG GGTTGCTATT TTCTAGAT 1620 TACTTTTTAG GGAACAAAATAAAATCCTTT GTT 1653 224 amino acids amino acid <Unknown> linear 2 MetPro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Al 1 5 10 15 AsnThr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Se 20 25 30 TrpGly Ile Leu Phe Ser His Pro Arg Asp Phe Thr Pro Val Cys Th 35 40 45 ThrGlu Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Ar 50 55 60 AsnVal Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Asp His Le 65 70 75 80Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Gl 85 90 95Lys Leu Pro Phe Pro Ile Ile Asp Asp Arg Asn Arg Glu Leu Ala Il 100 105110 Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pr 115120 125 Val Thr Ala Arg Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Ly130 135 140 Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp GluIl 145 150 155 160 Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu LysArg Val Al 165 170 175 Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val MetVal Leu Pro Th 180 185 190 Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe ProLys Gly Val Phe Th 195 200 205 Lys Glu Leu Pro Ser Gly Lys Lys Tyr LeuArg Tyr Thr Pro Gln Pr 210 215 220 1193 base pairs nucleic acid singlelinear 3 AGCGTCACCA CTGCCGCCAT GCCCGGAGGT TTGCTTCTCG GGGACGAAGCCCCCAACTTT 60 GAGGCCAATA CCACCATCGG CCGCATCCGC TTCCACGATT TCCTGGGAGATTCATGGGG 120 ATTCTCTTTT CCCACCCACG GGACTTTACC CCAGTGTGCA CCACAGAACTTGGCAGAGC 180 GCAAAGCTGG CGCCAGAGTT TGCCAAGAGG AATGTTAAGT TGATTGCTCTTTCAATAGA 240 AGTGTTGAGG ATCATCTTGC CTGGAGCAAG GACATCAATG CTTACAATGGTGAAACACC 300 ACGGAAAAGT TGCCATTTCC CATCATTGAT GATAAGGGCA GGGACCTTGCCATCCTTTT 360 GGCATGTTGG ATCCAGTCGA GAAGGACGCT AACAACATGC CTGTGACGGCCCGTGTGGT 420 TTCATTTTTG GCCCTGACAA GAAACTGAAG CTGTCTATCC TCTACCCTGCCACCACGGG 480 AGGAACTTTG ATGAGATTCT CAGAGTGGTA CAATGTTTCC CTAAAGGAGTCTTCACCAA 540 GAGCTCCCGT CTGGCAAAAA ATACCTCCGT TATACACCCC AGCCTTAAGTCTTTGCGGA 600 ATTGGGGCTG CATCTGCACA TCCAGTACTG GGGCCTGAGG ATGTCAGCTGGCAGCCGTG 660 GTCCTTGCAG CAGGTCCGTA GAAAGATCGT GGCATGATCA CAGCCGGTCCTGTAGATCG 720 TCGCTATACT ACTGGGTCAT TAAATGGAAA TGGCACCAAA ACCTTCTCGGGATTCTTTA 780 TCTGTGCCTT CGCCAGCATT CTGCCCCTCT GCCTGTCACA GTGCCCTACTGACTGGCTC 840 CTTTGAAACG AATTATGTAT TGAAGATTCC TTAGGTCTCT GTAGGGTCTTTGATCAGCA 900 ACAAGGTAGT GTCAGTGTGG GCTCTGTGCT AGAATGATGA AACACCTTTTGTATCTTTC 960 GAACTGAATC TTCTGTTACC CATTTTGGAG AGCACTGACA TAGGGAGAAGCTTTCGAT 1020 TGTATTTTTA GTAAATAAAA AGTGGGGACA GCCGGGAGAA TTCTTACAGGGAATCTAT 1080 TAAGTTTCTA TCGAAGTGGG CTCAGAAACC TTTCGCCTCC CAAGAGTGCGCATGTACC 1140 CTAGAGTTTC CACATCTGCT CTCTGGTGAT GTCTGCCTGT GAACGCACCT TAT1193 224 amino acids amino acid <Unknown> linear 4 Met Pro Gly Gly LeuLeu Leu Gly Asp Glu Ala Pro Asn Phe Glu Al 1 5 10 15 Asn Thr Thr Ile GlyArg Ile Arg Phe His Asp Phe Leu Gly Asp Se 20 25 30 Trp Gly Ile Leu PheSer His Pro Arg Asp Phe Thr Pro Val Cys Th 35 40 45 Thr Glu Leu Gly ArgAla Ala Lys Leu Ala Pro Glu Phe Ala Lys Ar 50 55 60 Asn Val Lys Leu IleAla Leu Ser Ile Asp Ser Val Glu Asp His Le 65 70 75 80 Ala Trp Ser LysAsp Ile Asn Ala Tyr Asn Gly Glu Thr Pro Thr Gl 85 90 95 Lys Leu Pro PhePro Ile Ile Asp Asp Lys Gly Arg Asp Leu Ala Il 100 105 110 Leu Leu GlyMet Leu Asp Pro Val Glu Lys Asp Ala Asn Asn Met Pr 115 120 125 Val ThrAla Arg Val Val Phe Ile Phe Gly Pro Asp Lys Lys Leu Ly 130 135 140 LeuSer Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Il 145 150 155160 Leu Arg Val Val Asp Ser Leu Gln Leu Thr Gly Thr Lys Pro Val Al 165170 175 Thr Pro Val Asp Trp Lys Lys Gly Glu Ser Val Met Val Val Pro Th180 185 190 Leu Ser Glu Glu Glu Ala Lys Gln Cys Phe Pro Lys Gly Val PheTh 195 200 205 Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr ProGln Pr 210 215 220 114 amino acids amino acid <Unknown> linear 5 His AspPhe Leu Gly Asp Ser Trp Gly Ile Leu Phe Ser His Pro Ar 1 5 10 15 Asp PheThr Pro Val Cys Thr Thr Glu Leu Gly Arg Ala Ala Lys Le 20 25 30 Ala ProGlu Phe Ala Lys Arg Asn Val Lys Leu Ile Ala Leu Ser Il 35 40 45 Asp SerVal Glu Asp His Leu Ala Trp Ser Lys Asp Ile Asn Ala Ty 50 55 60 Asn GlyGlu Thr Pro Thr Glu Leu Tyr Pro Ala Thr Thr Gly Arg As 65 70 75 80 PheAsp Glu Ile Leu Arg Val Val Asp Ser Leu Gln Leu Thr Gly Th 85 90 95 LysPro Val Ala Thr Pro Val Asp Trp Lys Lys Gly Glu Ser Val Me 100 105 110Val Val 114 amino acids amino acid <Unknown> linear 6 Leu Asp Asp PheLys Gly Lys Tyr Leu Val Leu Phe Phe Tyr Pro Le 1 5 10 15 Asp Phe Thr PheVal Cys Pro Thr Glu Ile Val Ala Phe Ser Asp Ly 20 25 30 Ala Asn Glu PheHis Asp Val Asn Cys Glu Val Val Ala Val Ser Va 35 40 45 Asp Ser His PheSer His Leu Ala Trp Ile Asn Thr Pro Arg Lys As 50 55 60 Gly Gly Leu GlyHis Met Asn Val Asn Asp Leu Pro Val Gly Arg Se 65 70 75 80 Val Glu GluThr Leu Arg Leu Val Lys Ala Phe Gln Phe Val Glu Th 85 90 95 His Gly GluVal Cys Pro Ala Asn Trp Thr Pro Glu Ser Pro Thr Il 100 105 110 Lys Pro114 amino acids amino acid <Unknown> linear 7 Leu Ser Glu Tyr Lys GlyLys Tyr Val Val Phe Phe Phe Tyr Pro Le 1 5 10 15 Asp Phe Thr Phe Val CysPro Thr Glu Ile Ile Ala Phe Ser Asp Ar 20 25 30 Ala Asp Glu Phe Lys LysLeu Asn Cys Gln Val Ile Gly Ala Ser Va 35 40 45 Asp Ser His Phe Cys HisLeu Ala Trp Ile Asn Thr Pro Lys Lys Gl 50 55 60 Gly Gly Leu Gly Pro MetAsn Ile Asn Asp Leu Pro Val Gly Arg Se 65 70 75 80 Val Asp Glu Ile IleArg Leu Val Gln Ala Phe Gln Phe Thr Asp Ly 85 90 95 His Gly Glu Val CysPro Ala Gly Trp Lys Pro Gly Ser Asp Thr Il 100 105 110 Lys Pro 114 aminoacids amino acid <Unknown> linear 8 Leu Ser Asp Tyr Arg Gly Lys Tyr ValVal Leu Phe Phe Tyr Pro Le 1 5 10 15 Asp Phe Thr Phe Val Cys Pro Thr GluIle Ile Ala Phe Ser Asp Hi 20 25 30 Ala Glu Asp Phe Arg Lys Leu Gly CysGlu Val Leu Gly Val Ser Va 35 40 45 Asp Ser Gln Phe Thr His Leu Ala TrpIle Asn Thr Pro Arg Lys Gl 50 55 60 Gly Gly Leu Ala Pro Leu Asn Val AsnAsp Leu Pro Val Gly Arg Se 65 70 75 80 Val Asp Glu Ala Leu Arg Leu ValGln Ala Phe Gln Tyr Thr Asp Gl 85 90 95 His Gly Glu Val Cys Pro Ala GlyTrp Lys Pro Gly Ser Asp Asn Il 100 105 110 Lys Pro 19 base pairs nucleicacid single linear 9 GCGAGCGATC TACAGGACC 19 20 base pairs nucleic acidsingle linear 10 TGATGGTAGT TCCCACCCTC 20 21 base pairs nucleic acidsingle linear 11 AGCGTCACCA CTGCCGCCAT G 21 23 base pairs nucleic acidsingle linear 12 GTACTGGATG TGCAGATGCA GCC 23

What is claimed is:
 1. An isolated polypeptide designated AOP2.
 2. Theisolated polypeptide of claim 1, wherein the polypeptide is a humanpolypeptide.
 3. The isolated polypeptide of claim 1, wherein thepolypeptide is a murine polypeptide.
 4. The isolated polypeptide ofclaim 2, wherein the polypeptide has the sequence set forth in SEQ IDNO:2.
 5. The isolated polypeptide of claim 3, wherein the polypeptidehas the sequence set forth in SEQ ID NO:4.
 6. An antigen compositioncomprising an Aop2 polypeptide or a fragment thereof and apharmaceutically acceptable buffer or diluent.
 7. The antigencomposition of claim 6, wherein the polypeptide is a human polypeptide.8. The antigen composition of claim 6, wherein the polypeptide is amurine polypeptide.
 9. The antigen composition of claim 7, wherein thepolypeptide has the sequence set forth in SEQ ID NO:2.
 10. The antigencomposition of claim 8, wherein the polypeptide has the sequence setforth in SEQ ID NO:4.
 11. A nucleic acid encoding an AOP2 polypeptide.12. The nucleic acid of claim 11, wherein the polypeptide is a humanpolypeptide.
 13. The nucleic acid of claim 11, wherein the polypeptideis a murine polypeptide.
 14. The nucleic acid of claim 12, wherein thepolypeptide has the sequence set forth in SEQ ID NO:2.
 15. The nucleicacid of claim 13, wherein the polypeptide has the sequence set forth inSEQ ID NO:4.
 16. A oligonucleotide comprising at least about 10consecutive bases of the nucleic acid sequence set forth in SEQ ID NO:1or SEQ ID NO:3.
 17. The oligonucleotide of claim 16, wherein saidoligonucleotide is at least about 15 consecutive bases of the nucleicacid set forth in SEQ ID NO:1 or SEQ ID NO:3.
 18. The oligonucleotide ofclaim 17, wherein said oligonucleotide is at least about 20 consecutivebases of the nucleic acid set forth in SEQ ID NO: 1 or SEQ ID NO:3. 19.The oligonucleotide of claim 18, wherein said oligonucleotide is atleast about 25 consecutive bases of the nucleic acid set forth in SEQ IDNO: 1 or SEQ ID NO:3.
 20. The oligonucleotide of claim 19, wherein saidoligonucleotide is at least about 30 consecutive bases of the nucleicacid set forth in SEQ ID NO: 1 or SEQ ID NO:
 3. 21. The oligonucleotideof claim 20, wherein said oligonucleotide is at least about 35consecutive bases of the nucleic acid set forth in SEQ ID NO: 1 or SEQID NO:3.
 22. The oligonucleotide of claim 21, wherein saidoligonucleotide is at least about 40 consecutive bases of the nucleicacid set forth in SEQ ID NO: 1 or SEQ ID NO:3.
 23. The oligonucleotideof claim 22, wherein said oligonucleotide is at least about 45consecutive bases of the nucleic acid set forth in SEQ ID NO:1 or SEQ IDNO:3.
 24. The oligonucleotide of claim 16, wherein said oligonucleotideis at least about 50 consecutive bases of the nucleic acid set forth inSEQ ID NO: 1 or SEQ ID NO:3.
 25. A method for diagnosing apredisposition to atherosclerotic lesions in a subject comprising: (i)obtaining a sample from said subject; and (ii) evaluating said samplefor the presence of an AOP2 polypeptide.
 26. The method of claim 25,wherein said sample is selected from the group consisting of heart,artery, vein, skin, muscle, facia, brain, prostate, breast, endometrium,lung, pancreas, small intestine, blood cells, liver, testes, ovaries,colon, skin, stomach, esophagus, spleen, lymph node, bone marrow orkidney, lymph fluid, ascites, serous fluid, pleural effusion, sputum,cerebrospinal fluid, lacrimal fluid, stool and urine.
 27. The method ofclaim 25, wherein said subject is a human.
 28. The method of claim 25,wherein said evaluating comprises determining the antioxidant activityof an AOP2 polypeptide of said sample.
 29. The method of claim 25,wherein said evaluating comprises determining the level of an AOP2polypeptide in cells of said sample.
 30. The method of claim 29, whereinsaid determining comprises quantitative PCR.
 31. The method of claim 29,wherein said determining comprises contacting said sample with anantibody that binds immunologically to an AOP2 polypeptide.
 32. Themethod of claim 25, wherein said evaluating comprises determining thesequence of a nucleic acid from said sample that encodes an AOP2polypeptide.
 33. A method for screening a compound for AOP2 stimulatoryactivity comprising: (i) providing an AOP2 polypeptide havingantioxidant activity; (ii) contacting said AOP2 polypeptide with acandidate stimulator; and (iii) determining the antioxidant activity ofsaid AOP2 polypeptide in the presence and absence of said candidatestimulator.
 34. A method for screening a compound for antioxidantstimulatory activity comprising: (i) providing a cell comprising annucleic acid encoding an active AOP2 polypeptide; (ii) contacting saidcell with a candidate stimulator; and (iii) determining the antioxidantactivity in said cell in the presence and absence of said candidatestimulator.
 35. The method of claim 34, wherein said cell is located ina non-human animal.
 36. A method for screening a compound foranti-atherosclerotic activity comprising: (i) providing a lipid; (ii)contacting said lipid with a candidate antioxidant; and (iii)determining the oxidation state of said lipid.
 37. A monoclonal antibodythat binds immunologically to an AOP2 polypeptide.
 38. A polyclonalantisera, antibodies of which bind immunologically to an AOP2polypeptide.
 39. An expression vector comprising a nucleic acid encodingan AOP2 polypeptide, said nucleic acid positioned in operable relationto a promoter.
 40. A recombinant host cell comprising a nucleic acidencoding an AOP2 polypeptide, said nucleic acid positioned in operablerelation to a promoter.
 41. A method for increasing AOP2 function in acell comprising: (i) providing a nucleic acid encoding an AOP2polypeptide having antioxidant activity, said nucleic acid positioned inoperable relation to a promoter; and (ii) contacting said nucleic acidwith said cell under conditions permitting the uptake of said nucleicacid.
 42. The method of claim 41, wherein said AOP2 polypeptide is ahuman polypeptide.
 43. The method of claim 42, wherein said AOP2polypeptide has the sequence set forth in SEQ ID NO:2.
 44. The method ofclaim 41, wherein said nucleic acid further comprises an expressionvector.
 45. The method of claim 44, wherein said expression vector isencapsulated in a liposome.
 46. The method of claim 44, wherein saidexpression vector is a viral vector.
 47. The method of claim 46, whereinsaid viral vector is selected from the group consisting of an adenoviralvector, a retroviral vector, a vaccinia viral vector, anadeno-associated viral vector or a herpesviral vector.
 48. The method ofclaim 41, wherein said cell is located in a human subject.
 49. Themethod of claim 41, wherein said cell is located in an experimentalanimal.
 50. The method of claim 49, wherein said nucleic acid isadministered intravenously.
 51. The method of claim 41, wherein saidpromoter is selected from the group consisting of CMV, RSV and E1A. 52.A method of reducing atherosclerotic lesions in a subject comprisingadministering to said subject a lipid antioxidant composition.